Alteration of FcRn binding affinities or serum half-lives of antibodies by mutagenesis

ABSTRACT

The present invention provides for a modified antibody of class IgG, in which at least one amino acid from the heavy chain constant region selected from the group consisting of amino acid residues 250, 314, and 428 is substituted with another amino acid which is different from that present in the unmodified antibody, thereby altering the binding affinity for FcRn and/or the serum half-life in comparison to the unmodified antibody.

RELATED APPLICATIONS

This application is a continuation-in-part of U.S. Ser. No. 10/822,300filed Apr. 9, 2004, which is a continuation-in-part of U.S. Ser. No.10/687,118 filed Oct. 15, 2003, which claims priority from U.S.Provisional Application Nos. 60/418,972 filed Oct. 15, 2002, 60/462,014filed Apr. 10, 2003, 60/475,762 filed Jun. 3, 2003, and 60/499,048 filedAug. 29, 2003, each of which is hereby incorporated by reference in itsentirety.

FIELD OF THE INVENTION

The present invention relates to the fields of immunology and proteinengineering. In particular, it concerns modified antibodies of class IgGthat have altered binding affinities for FcRn, or altered serumhalf-lives as a consequence of one or more amino acid modifications inthe Fc region thereof.

BACKGROUND OF THE INVENTION

Antibodies are proteins that exhibit binding specificity to a particularantigen. Native (i.e., naturally occurring or wild-type) antibodies areusually heterotetrameric glycoproteins of about 150,000 daltons,composed of two identical light (L) chains and two identical heavy (H)chains. As shown in FIG. 1, each light chain is linked to a heavy chainby one covalent disulfide bond, while the number of disulfide linkagesvaries between the heavy chains of different immunoglobulin isotypes.Each heavy chain has at one end a variable domain (V_(H)) followed by anumber of constant domains. Each light chain has a variable domain(V_(L)) at one end and a constant domain at the other end. The constantdomain of the light chain is aligned with the first constant domain ofthe heavy chain.

Certain portions of the variable domains differ extensively in sequenceamong antibodies and are responsible for the binding specificity of eachparticular antibody to its particular antigen. The constant domains arenot involved directly in binding of an antibody to an antigen, butexhibit various effector functions. Depending on the amino acid sequenceof the constant region of the heavy chains, antibodies orimmunoglobulins can be assigned to different classes. There are fivemajor classes (isotypes) of immunoglobulins in humans: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (subtypes), such as IgG1, IgG2, IgG3, and IgG4 as well asIgA1 and IgA2.

A schematic representation of the native IgG structure is shown in FIG.1, where the various portions of the native antibody molecule areindicated. The heavy chain constant region includes C_(H)1, the hingeregion, C_(H)2, and C_(H)3. Papain digestion of antibodies produces twofragments, Fab and Fc. The Fc fragment consists of C_(H)2, C_(H)3, andpart of the hinge region. The crystal structure of the human IgG1 Fcfragment has been determined (Deisenhofer, Biochemistry 20:2361-2370(1981)). In human IgG molecules, the Fc fragment is generated by papaincleavage of the hinge region N-terminal to Cys 226. Therefore, the humanIgG heavy chain Fc region is usually defined as stretching from theamino acid residue at position 226 to the C-terminus (numbered accordingto the EU index of Kabat, et al., “Sequences of Proteins ofImmunological Interest”, 5^(th) ed., National Institutes of Health,Bethesda, Md. (1991); the EU numbering scheme is used hereinafter).

The Fc region is essential to the effector functions of antibodies. Theeffector functions include initiating complement-dependent cytotoxicity(CDC), initiating phagocytosis and antibody-dependent cell-mediatedcytotoxicity (ADCC), and transferring antibodies across cellularbarriers by transcytosis. In addition, the Fc region is critical formaintaining the serum half-life of an antibody of class IgG (Ward andGhetie, Ther. Immunol. 2:77-94 (1995)).

Studies have found that the serum half-life of an IgG antibody ismediated by binding of Fc to the neonatal Fc receptor (FcRn). FcRn is aheterodimer consisting of a transmembrane α chain and a soluble β chain(β2-microglobulin). FcRn shares 22-29% sequence identity with Class IMHC molecules and has a non-functional version of the MHCpeptide-binding groove (Simister and Mostov, Nature 337:184-187 (1989)).The α1 and α2 domains of FcRn interact with the C_(H)2 and C_(H)3domains of the Fc region (Raghavan et al., Immunity 1:303-315 (1994)).

A model has been proposed for how FcRn might regulate the serumhalf-life of an antibody. As shown in FIG. 2, IgGs are taken up byendothelial cells through non-specific pinocytosis and then enter acidicendosomes. FcRn binds IgG at acidic pH (<6.5) in endosomes and releasesIgG at basic pH (>7.4) in the bloodstream. Accordingly, FcRn salvagesIgG from a lysosomal degradation pathway. When serum IgG levelsdecrease, more FcRn molecules are available for IgG binding so that anincreased amount of IgG is salvaged. Conversely, if serum IgG levelsrise, FcRn becomes saturated, thereby increasing the proportion ofpinocytosed IgG that is degraded (Ghetie and Ward, Annu. Rev. Immunol.18:739-766 (2000)).

Consistent with the above model, the results of numerous studies supporta correlation between the affinity for FcRn binding and the serumhalf-life of an antibody (Ghetie and Ward, ibid.). Significantly, such acorrelation has been extended to engineered antibodies with higheraffinity for FcRn than their wild-type parent molecules.

Ghetie et al. randomly mutagenized position 252, position 254, andposition 256 in a mouse IgG1 Fc-hinge fragment. One mutant showed anaffinity three and a half times higher for mouse FcRn and a half-lifeabout 23% or 65% longer in two mouse strains, respectively, as comparedto that of the wild-type (Ghetie et al., Nat. Biotechnol. 15:637-640(1997)).

Shields et al. used alanine scanning mutagenesis to alter residues inthe Fc region of a human IgG1 antibody and then assessed the binding tohuman FcRn. They found several mutants with a higher binding affinityfor human FcRn than the wild-type, but did not identify mutations atpositions 250, 314, or 428 (Shields et al., J. Biol. Chem. 276:6591-6604(2001)).

Martin et al. proposed mutagenesis at a number of positions in the humanIgG Fc to increase binding to FcRn including, among many others,positions 250, 314, and 428. However, none of the mutants proposed byMartin et al. was constructed or tested for binding to FcRn (Martin etal., Mol. Cell 7:867-877 (2001)).

Dall'Acqua et al. described random mutagenesis and screening of humanIgG1 hinge-Fc fragment phage display libraries against mouse FcRn. Theydisclosed random mutagenesis of positions 428-436 but did not identifymutagenesis at position 428 as having any effect on mouse FcRn bindingaffinity and stated that the wild-type methionine amino acid at thisposition is favorable for efficient binding (Dall'Acqua et al., J.Immunol. 169:5171-5180 (2002)).

Kim et al. mutagenized human IgG1 by amino acid substitutions atposition 253, position 310, or position 435 of the Fc region. They foundthat the mutant Fc-hinge fragments have reduced serum half-lives in micecompared to the wild-type IgG1 Fc-hinge fragment, and concluded thatIle253, His310, and His435 play a central role in regulating the serumhalf-life of IgG (Kim et al., Eur. J. Immunol. 29:2819-2825 (1999)).

Hornick et al. showed that a single amino acid substitution at position253 in the Fc region of a chimeric human IgG1 antibody acceleratesclearance in mice and improves immunoscintigraphy of solid tumors(Hornick et al., J. Nucl. Med. 41:355-362 (2000)).

U.S. Pat. No. 6,165,745 discloses a method of producing an antibody witha decreased biological half-life by introducing a mutation into the DNAsegment encoding the antibody. The mutation includes an amino acidsubstitution at position 253, 310, 311, 433, or 434 of the Fc-hingedomain. The full disclosure of U.S. Pat. No. 6,165,745, as well as thefull disclosure of all other U.S. patent references cited herein, arehereby incorporated by reference.

U.S. Pat. Nos. 5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370disclose the humanization of immunoglobulins.

U.S. Pat. No. 6,277,375 B1 discloses a composition comprising a mutantIgG molecule having an increased serum half-life relative to thewild-type IgG, wherein the mutant IgG molecule comprises the amino acidsubstitutions: threonine to leucine at position 252, threonine to serineat position 254, or threonine to phenylalanine at position 256. A mutantIgG with an amino acid substitution at position 433, 435, or 436 is alsodisclosed.

U.S. Patent Application No. 20020098193 A1 and PCT Publication No. WO97/34621 disclose mutant IgG molecules having increased serum half-livesrelative to IgG wherein the mutant IgG molecule has at least one aminoacid substitution in the Fc-hinge region. However, no experimentalsupport is provided for mutations at positions 250, 314, or 428.

U.S. Pat. No. 6,528,624 discloses a variant of an antibody comprising ahuman IgG Fc region, which variant comprises an amino acid substitutionat one or more of amino acid positions 270, 322, 326, 327, 329, 331,333, and 334 of the human IgG Fc region.

PCT Publication No. WO 98/05787 discloses deleting or substituting aminoacids at positions 310-331 of the BR96 antibody in order to reduce itsinduced toxicity, but does not disclose amino acid modifications thatresult in altered binding to FcRn.

PCT Publication No. WO 00/42072 discloses a polypeptide comprising avariant Fc region with altered FcRn binding affinity, which polypeptidecomprises an amino acid modification at any one or more of amino acidpositions 238, 252, 253, 254, 255, 256, 265, 272, 286, 288, 303, 305,307, 309, 311, 312, 317, 340, 356, 360, 362, 376, 378, 380, 386, 388,400, 413, 415, 424, 433, 434, 435, 436, 439, and 447 of the Fc region,wherein the numbering of the residues in the Fc region is that of the EUindex (Kabat et al., op. cit.).

PCT Publication No. WO 02/060919 A2 discloses a modified IgG comprisingan IgG constant domain comprising one or more amino acid modificationsrelative to a wild-type IgG constant domain, wherein the modified IgGhas an increased half-life compared to the half-life of an IgG havingthe wild-type IgG constant domain, and wherein the one or more aminoacid modifications are at one or more of positions 251, 253, 255,285-290, 308-314, 385-389, and 428-435. However, no examples ofmutations at positions 314 or 428 with altered binding to FcRn aredisclosed.

Martin, W. L. (Doctoral dissertation entitled, “Protein-ProteinRecognition: The Neonatal Fc Receptor and Immunoglobulin G,” CaliforniaInstitute of Technology (2001)) proposes theoretical mutations atseveral Fc positions of the rat gamma-2a constant region, includingpositions 250 and 428, that may increase FcRn binding affinity. Martinsuggests the possibility of substituting, among others, isoleucine forvaline at position 250, or substituting phenylalanine for leucine atposition 428. Martin does not suggest any substitution for position 314.Martin does not demonstrate increased binding affinity to FcRn for anyof these proposed mutations.

The above-referenced publications have not showed that the serumhalf-life or FcRn binding affinity of an antibody of the IgG class canbe altered by the amino acid modifications at position 250, position314, or position 428 of the Fc region. The present invention usedmolecular modeling to select Fc residues near the FcRn contact site thatmight have an effect on binding, but may not be necessary forpH-dependent binding. Amino acid modifications were made at position250, 314, or 428 of the constant region of an immunoglobulin heavy chainof class IgG. The serum half-lives or FcRn binding affinities ofantibodies comprising said modifications were altered and, therefore,were different from those of unmodified antibodies.

SUMMARY OF THE INVENTION

The present invention is based upon the inventors' identification ofseveral mutations in the constant domain of a human IgG molecule thatalter (i.e., increase or decrease) the affinity of the IgG molecule forFcRn. The present invention provides for modified antibodies havingaltered FcRn binding affinity and/or serum half-life relative to thecorresponding unmodified antibody. The in vivo half-life (i.e.persistence in serum or other tissues of a subject) of antibodies, andother bioactive molecules, is an important clinical parameter thatdetermines the amount and frequency of antibody (or any otherpharmaceutical molecule) administration. Accordingly, such molecules,including antibodies, with increased (or decreased) half-life are ofsignificant pharmaceutical importance.

The present invention relates to a modified molecule (preferably anantibody), that has an increased (or decreased) in vivo half-life byvirtue of the presence of a modified IgG constant domain (preferablyfrom a human IgG), or FcRn-binding portion thereof (preferably the Fc orhinge-Fc domain) wherein the IgG constant domain, or fragment thereof,is modified (preferably by an amino acid substitution) to increase (ordecrease) the affinity for the FcRn.

In a particular embodiment, the present invention relates to modifiedclass IgG antibodies, whose in vivo half-lives are extended (or reduced)by the changes in amino acid residues at positions identified bystructural studies to be involved either directly or indirectly in theinteraction of the hinge-Fc domain with the FcRn receptor. In apreferred embodiment the modified class IgG antibody is selected fromthe group consisting of daclizumab, fontolizumab, visilizumab and M200(volociximab).

In preferred embodiments, the constant domain (or fragment thereof) hasa higher affinity for FcRn at pH 6.0 than at pH 7.4. That is, the pHdependency of FcRn binding affinity mimics the wild-type pH dependency.In alternative embodiments, the modified antibodies of the presentinvention may exhibit altered pH dependence profiles relative to that ofthe unmodified antibody. Such altered pH dependence profiles may beuseful in some therapeutic or diagnostic applications.

In some embodiments, the antibody modifications of the present inventionwill alter FcRn binding and/or serum half-life without altering otherantibody effector functions such as ADCC or CDC. In particularlypreferred embodiments, the modified antibodies of the invention exhibitno changes in binding to Fc-gamma receptors or C1q. In alternativeembodiments, the antibody modifications of the present invention mayresult in increased (or decreased) effector functions as well asincreased serum half-life. In particularly preferred embodiments, themodified antibodies of the invention may have increased (or decreased)ADCC activities as well as increased serum half-life.

It should be noted that the modifications of the present invention mayalso alter (i.e., increase or decrease) the bioavailability (e.g.,transport to mucosal surfaces, or other target tissues) of the modifiedantibodies (or other molecules).

In preferred embodiments, the present invention provides for a modifiedantibody of class IgG, in which at least one amino acid from the heavychain constant region selected from the group consisting of amino acidresidues 250, 314, and 428 is substituted with an amino acid residuedifferent from that present in the unmodified antibody. Preferably thissubstitution alters the binding affinity for FcRn and/or the serumhalf-life of said modified antibody relative to the unmodified wild-typeantibody. The present invention further provides for a modified antibodyhaving an increased binding affinity for FcRn and an increased serumhalf-life as compared with the unmodified antibody, wherein amino acidresidue 250 from the heavy chain constant region is substituted withglutamic acid or glutamine; or amino acid residue 428 from the heavychain constant region is substituted with phenylalanine or leucine.

The present invention further provides for a modified antibody having anincreased binding affinity for FcRn and/or an increased serum half-lifeas compared with the unmodified antibody, wherein (a) amino acid residue250 from the heavy chain constant region is substituted with glutamicacid and amino acid residue 428 from the heavy chain constant region issubstituted with phenylalanine; (b) amino acid residue 250 from theheavy chain constant region is substituted with glutamine and amino acidresidue 428 from the heavy chain constant region is substituted withphenylalanine; or (c) amino acid residue 250 from the heavy chainconstant region is substituted with glutamine and amino acid residue 428from the heavy chain constant region is substituted with leucine.

The present invention further provides for a modified antibody having areduced binding affinity for FcRn and/or a reduced serum half-life ascompared with the unmodified antibody, wherein amino acid residue 314from the heavy chain constant region is substituted with another aminoacid which is different from that present in an unmodified antibody.

The present invention further provides for a modified antibody having areduced binding affinity for FcRn and/or a reduced serum half-life ascompared with the unmodified antibody, wherein amino acid residue 250from the heavy chain constant region is substituted with arginine,asparagine, aspartic acid, lysine, phenylalanine, proline, tryptophan,or tyrosine; or amino acid residue 428 from the heavy chain constantregion is substituted with alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, lysine, proline,serine, threonine, tyrosine, or valine.

The present invention also provides for an antibody having a constantregion substantially identical to a naturally occurring class IgGantibody constant region wherein at least one amino acid residueselected from the group consisting of residues 250, 314, and 428 isdifferent from that present in the naturally occurring class IgGantibody, thereby altering FcRn binding affinity and/or serum half-lifeof said antibody relative to the naturally occurring antibody. Inpreferred embodiments, the naturally occurring class IgG antibodycomprises a heavy chain constant region of a human IgG1, IgG2, IgG2M3,IgG3 or IgG4 molecule. Also in preferred embodiments, amino acid residue250 from the heavy chain constant region of the antibody having aconstant region substantially identical to the naturally occurring classIgG antibody is glutamic acid or glutamine; or amino acid residue 428from the heavy chain constant region is phenylalanine or leucine. Inother preferred embodiments, the antibody having a constant regionsubstantially identical to a naturally occurring class IgG antibody hasa glutamic acid residue at position 250 and, phenylalanine residue atposition 428; or amino acid residue 250 is glutamine and amino acidresidue 428 is phenylalanine; or amino acid residue 250 is glutamine andamino acid residue 428 is leucine.

In some embodiments, the antibody having a constant region substantiallyidentical to a naturally occurring class IgG antibody constant regionincludes an amino acid residue at position 314 different from thatpresent in the naturally occurring antibody, thereby reducing FcRnbinding affinity and/or reducing serum half-life relative to thenaturally occurring antibody. Embodiments include antibodies whereinamino acid residue 314 is alanine, arginine, aspartic acid, asparagine,cysteine, glutamic acid, glutamine, glycine, histidine, lysine,methionine, phenylalanine, proline, serine, threonine, tryptophan,tyrosine, or valine. In one preferred embodiment amino acid residue 314is arginine.

In other embodiments, the antibody having a constant regionsubstantially identical to a naturally occurring class IgG antibodyconstant region includes an amino acid residue at position 250 selectedfrom the group consisting of arginine, asparagine, aspartic acid,lysine, phenylalanine, proline, tryptophan, or tyrosine, therebyreducing FcRn binding affinity and/or reducing serum half-life relativeto the naturally occurring antibody. Similarly, the amino acid residueat position 428 may be substituted with an amino acid residue selectedfrom the group consisting of alanine, arginine, asparagine, asparticacid, cysteine, glutamic acid, glutamine, glycine, histidine, lysine,proline, serine, threonine, tyrosine, or valine, thereby reducing FcRnbinding affinity and/or reducing serum half-life relative to thenaturally occurring antibody.

The present invention further provides for a method of modifying anantibody of class IgG, wherein said method comprises substituting atleast one amino acid from the heavy chain constant region selected fromthe group consisting of amino acid residues 250, 314, and 428 with anamino acid which is different from that present in an unmodifiedantibody, thereby causing an alteration of the binding affinity for FcRnand/or the serum half-life of said unmodified antibody.

The present invention further provides for a method of producing amodified antibody of class IgG with an altered binding affinity for FcRnand/or an altered serum half-life as compared with an unmodifiedantibody, wherein said method comprises:

(a) preparing an expression vector (preferably a replicable expressionvector) comprising a suitable promoter operably linked to DNA encodingat least a constant region of an immunoglobulin heavy chain wherein atleast one amino acid from the heavy chain constant region selected fromthe group consisting of amino acid residues 250, 314, and 428 issubstituted with an amino acid which is different from that present inan unmodified antibody thereby causing an alteration in FcRn bindingaffinity and/or serum half-life;

(b) transforming host cells with said vector; and

(c) culturing said transformed host cells to produce said modifiedantibody.

Optionally, such a method further comprises: preparing a secondexpression vector (preferably a replicable expression vector) comprisinga promoter operably linked to DNA encoding a complementaryimmunoglobulin light chain and further transforming said cell line withsaid second vector.

The present invention also includes pharmaceutical compositions andmethods of prophylaxis and therapy using modified immunoglobulins(including immunoglobulins conjugated with toxins and radionuclides),proteins and other bioactive molecules of the invention having alteredhalf-lives. Also included are methods of diagnosis using modifiedimmunoglobulins, proteins and other bioactive molecules of the inventionhaving altered half-lives. In preferred embodiments, the amino acidmodifications of the present invention may be used to extend the serumhalf-life of a therapeutic or diagnostic antibody. For example, thepresent invention provides for a modified therapeutic or diagnosticantibody of class IgG with an in vivo elimination half-life at leastabout 1.3-fold longer than that of the corresponding unmodifiedantibody. The modified therapeutic or diagnostic antibody wherein atleast one amino acid residue selected from the group consisting ofresidues 250, 314, and 428 is different from that present in theunmodified antibody. In preferred embodiments the modified therapeuticor diagnostic antibody has an in vivo elimination half-life at leastabout 1.3-fold, 1.5-fold, 1.8-fold, 1.9-fold, or greater than 2.0-foldlonger than that of the corresponding unmodified antibody.

The present invention also provides for a modified therapeutic ordiagnostic antibody of class IgG with an in vivo clearance at leastabout 1.3-fold lower than that of the corresponding unmodified antibody.The modified therapeutic or diagnostic antibody wherein at least oneamino acid residue selected from the group consisting of residues 250,314, and 428 is different from that present in the unmodified antibody.In preferred embodiments the modified therapeutic or diagnostic antibodyhas an in vivo clearance at least about 1.3-fold, 1.5-fold, 1.8-fold,2.0-fold, 2.3-fold, 2.5-fold, 2.8-fold, or greater than 3.0-fold lowerthan that of the corresponding unmodified antibody. In a preferredembodiment the therapeutic antibody is selected from the groupconsisting of daclizumab, fontolizumab, visilizumab and volociximab.

The present invention further provides for a modified therapeutic ordiagnostic antibody of class IgG with an in vivo area under theconcentration-time curve at least about 1.3-fold higher than that of thecorresponding unmodified antibody. The modified therapeutic ordiagnostic antibody wherein at least one amino acid residue selectedfrom the group consisting of residues 250, 314, and 428 is differentfrom that present in the unmodified antibody. In preferred embodimentsthe modified therapeutic or diagnostic antibody has an in vivoelimination half-life at least about 1.3-fold, 1.5-fold, 1.8-fold,2.0-fold, 2.3-fold, 2.6-fold, 2.8-fold, or greater than 3.0-fold higherthan that of the corresponding unmodified antibody.

In alternative preferred embodiments, the amino acid modifications ofthe present invention may also be used to reduce the serum half-life ofa therapeutic or diagnostic antibody. Such therapeutic or diagnosticantibodies are well-known in the art and listed in the followingdescription of the invention.

The invention also provides a modified therapeutic antibody comprising alight chain amino acid sequence of SEQ ID NO: 118 and a heavy chainamino acid sequence selected from SEQ ID NOs: 119-128. The inventionalso provides a vector comprising a polynucleotide encoding one or moreof these light or heavy chain amino acid sequences. This inventionfurther provides a host cell comprising this vector.

In addition the invention provides: a modified therapeutic antibodycomprising a light chain amino acid sequence of SEQ ID NO: 129 and aheavy chain amino acid sequence selected from SEQ ID NOs: 130-134; amodified therapeutic antibody comprising a light chain amino acidsequence of SEQ ID NO: 135 and a heavy chain amino acid sequenceselected from SEQ ID NOs: 136-140; and a modified therapeutic antibodycomprising a light chain amino acid sequence of SEQ ID NO: 141 and aheavy chain amino acid sequence selected from SEQ ID NOs: 142-146. Theinvention also provides vectors comprising one or more of the heavyand/or light chain amino acid sequences of the above-listed modifiedtherapeutic antibodies; and the invention provides a host cellcomprising any of the above listed vectors.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Illustration of the Structure of an IgG Molecule

FIG. 2. Salvage Pathway of IgG Molecules

FIG. 3. Amino Acid Sequences of Hu1D10-IgG2M3, Hu1D10-IgG1, Hu1D10-IgG3and Hu1D10-IgG4 with Positions 250, 314, and 428 of the Heavy ChainHighlighted. “Hu1D10-VH” (SEQ ID NO: 6) depicts the amino acid sequenceof the heavy chain variable region of Hu1D10-IgG2M3, Hu1D10-IgG1,Hu1D10-IgG3, or Hu1D10-IgG4. “IgG2M3-CH” (SEQ ID NO: 2) depicts theamino acid sequence of the heavy chain constant region of Hu1D10-IgG2M3.“IgG1-CH” (SEQ ID NO: 7) depicts the amino acid sequence of the heavychain constant region of Hu1D10-IgG1. “IgG3-CH” (SEQ ID NO: 113) depictsthe amino acid sequence of the heavy chain constant region ofHu1D10-IgG3. “IgG4-CH” (SEQ ID NO: 114) depicts the amino acid sequenceof the heavy chain constant region of Hu1D10-IgG4. “Hu1D10-VL” (SEQ IDNO: 8) depicts the amino acid sequence of the light chain variableregion of Hu1D10-IgG2M3, Hu1D10-IgG1, Hu1D10-IgG3, or Hu D10-IgG4.“KAPPA-CL” (SEQ ID NO: 9) depicts the amino acid sequence of the lightchain constant region of Hu1D10-IgG2M3, Hu1D10-IgG1, Hu1D10-IgG3, orHu1D10-IgG4.

FIG. 4. Illustration of the Overlap-Extension PCR Method

FIG. 5A. Restriction Map of Heavy Chain Vector pVAg2M3-OST577

FIG. 5B. Restriction Map of Heavy Chain Vector pVAg1.N-OST577

FIG. 6. Restriction Map of Light Chain Vector pVAλ2-OST577

FIG. 7A. Restriction Map of Heavy Chain Vector pVAg2M3-Hu1D10

FIG. 7B. Restriction Map of Heavy Chain Vector pVAg1.N-Hu1D10

FIG. 7C. Restriction Map of Heavy Chain Vector pHuHCg3.Tt.D-Hu1D10

FIG. 7D. Restriction Map of Heavy Chain Vector pHuHCg4.Tt.D-Hu1D10

FIG. 8. Restriction Map of Light Chain Vector pVk-Hu1D10.

FIG. 9A. Restriction Map of Human FcRn Vector pDL208

FIG. 9B. Restriction Map of Rhesus FcRn Vector pDL410

FIG. 10A. SDS-PAGE Analysis of OST577-IgG2M3 Wild-Type and MutantAntibodies Purified OST577-IgG2M3 wild-type and mutant antibodies wereanalyzed by SDS-PAGE under reducing conditions, as described in Example5.

FIG. 10B. SDS-PAGE Analysis of OST577-IgG1 Wild-Type and MutantAntibodies Purified OST577-IgG1 wild-type and mutant antibodies wereanalyzed by SDS-PAGE under reducing conditions, as described in Example5.

FIG. 11A. Single Point Competitive Binding Assay of the Various Mutantsof Position 250 of OST577-IgG2M3 to Human FcRn. The binding ofbiotinylated OST577-IgG2M3 antibody to human FcRn on transfected NS0cells in the presence of the wild-type or position 250 mutantOST577-IgG2M3 competitor antibodies in FBB, at pH 6.0, was detected withstreptavidin-conjugated RPE and analyzed by flow cytometry, as describedin Example 6.

FIG. 11B. Single Point Competitive Binding Assay of the Various Mutantsof Position 314 of OST577-IgG2M3 to Human FcRn. The binding ofbiotinylated OST577-IgG2M3 antibody to human FcRn on transfected NS0cells in the presence of the wild-type or position 314 mutantOST577-IgG2M3 competitor antibodies in FBB, at pH 6.0, was detected withstreptavidin-conjugated RPE and analyzed by flow cytometry, as describedin Example 6.

FIG. 11C. Single Point Competitive Binding Assay of the Various Mutantsof Position 428 of OST577-IgG2M3 to Human FcRn. The binding ofbiotinylated OST577-IgG2M3 antibody to human FcRn on transfected NS0cells in the presence of the wild-type or position 428 mutantOST577-IgG2M3 competitor antibodies in FBB, at pH 6.0, was detected withstreptavidin-conjugated RPE and analyzed by flow cytometry, as describedin Example 6.

FIG. 12A. Competitive Binding Assay of OST577-IgG2M3 Wild-Type andMutant Antibodies to Human FcRn. The binding of biotinylatedHuEP5C7-IgG2M3 antibody to human FcRn on transfected NS0 cells in thepresence of increasing concentrations of the wild-type or mutantOST577-IgG2M3 competitor antibodies in FBB, at pH 6.0, was detected withstreptavidin-conjugated RPE and analyzed by flow cytometry, as describedin Example 6.

FIG. 12B. Competitive Binding Assay of OST577-IgG2M3 Wild-Type andMutant Antibodies to Human FcRn. The binding of biotinylatedOST577-IgG2M3 antibody to human FcRn on transfected NS0 cells in thepresence of increasing concentrations of the wild-type or mutantOST577-IgG2M3 competitor antibodies in FBB, at pH 6.0, was detected withstreptavidin-conjugated RPE and analyzed by flow cytometry, as describedin Example 6.

FIG. 13. Antibody Binding to Cells Transfected with Human FcRn versusUntransfected Cells. The binding of the wild-type or mutantOST577-IgG2M3 antibodies to human FcRn on transfected NS0 cells or tountransfected NS0 cells in FBB, at pH 6.0, was analyzed by flowcytometry, as described in Example 7.

FIG. 14. Competitive Binding Assay of OST577-IgG2M3 Wild-Type and MutantAntibodies to Human FcRn at 37° C. The binding of biotinylatedOST577-IgG2M3 antibody to human FcRn on transfected NS0 cells in thepresence of increasing concentrations of the wild-type or mutantOST577-IgG2M3 competitor antibodies in FBB, at pH 6.0, was detected withstreptavidin-conjugated RPE and analyzed by flow cytometry, as describedin Example 7. All incubations were done at 37° C.

FIG. 15A. pH-Dependent Binding and Release of OST577-IgG2M3 Wild-Typeand Mutant Antibodies to Human FcRn. The binding and release of thewild-type or mutant OST577-IgG2M3 antibodies to human FcRn ontransfected NS0 cells in FBB, at pH 6.0, 6.5, 7.0, 7.5, or 8.0, wasanalyzed by flow cytometry, as described in Example 7.

FIG. 15B. pH-Dependent Binding and Release of OST577-IgG1 Wild-Type andMutant Antibodies to Human FcRn. The binding and release of thewild-type or mutant OST577-IgG1 antibodies to human FcRn on transfectedNS0 cells in FBB, at pH 6.0, 6.5, 7.0, 7.5, or 8.0, was analyzed by flowcytometry, as described in Example 7.

FIG. 15C. pH-Dependent Binding and Release of OST577-IgG1 Wild-Type andMutant Antibodies to Human FcRn. The binding and release of thewild-type or mutant OST577-IgG1 antibodies to human FcRn on transfectedNS0 cells in FBB, at pH 6.0, 6.5, 7.0, 7.5, or 8.0, was analyzed by flowcytometry, as described in Example 7.

FIG. 15D. pH-Dependent Binding and Release of OST577-IgG2M3 Wild-Typeand Mutant Antibodies to Rhesus FcRn. The binding and release of thewild-type or mutant OST577-IgG2M3 antibodies to rhesus FcRn ontransfected NS0 cells in FBB, at pH 6.0, 6.5, 7.0, 7.5, or 8.0, wasanalyzed by flow cytometry, as described in Example 7.

FIG. 15E. pH-Dependent Binding and Release of OST577-IgG1 Wild-Type andMutant Antibodies to Rhesus FcRn. The binding and release of thewild-type or mutant OST577-IgG1 antibodies to rhesus FcRn on transfectedNS0 cells in FBB, at pH 6.0, 6.5, 7.0, 7.5, or 8.0, was analyzed by flowcytometry, as described in Example 7.

FIG. 16A. Competitive Binding Assay of OST577-IgG2M3 Wild-Type andMutant Antibodies to HbsAg. The binding of the wild-type or mutantOST577-IgG2M3 antibodies to HBsAg was analyzed in an ELISA competition,as described in Example 8.

FIG. 16B. Competitive Binding Assay of OST577-IgG1 Wild-Type and MutantAntibodies to HbsAg. The binding of the wild-type or mutant OST577-IgG1antibodies to HBsAg was analyzed in an ELISA competition, as describedin Example 8.

FIG. 17A. Binding Assay of Hu1D10-IgG2M3 Wild-Type and Mutant Antibodiesto HLA-DR β Chain Allele. The binding of the wild-type or mutantHu1D10-IgG2M3 antibodies to Raji cells was analyzed in a FACS bindingassay, as described in Example 8.

FIG. 17B. Binding Assay of Hu1D10-IgG1 Wild-Type and Mutant Antibodiesto HLA-DR β Chain Allele. The binding of the wild-type or mutantHu1D10-IgG1 antibodies to Raji cells was analyzed in a FACS bindingassay, as described in Example 8.

FIG. 18A. ADCC Assay of Hu1D10-IgG1 and Hu1D10-IgG2M3 Wild-Type andMutant Antibodies Using PBMC From a 158V/V Donor. The ADCC activity ofthe wild-type or mutant Hu1D10-IgG1 and Hu1D10-IgG2M3 antibodies on Rajicells was determined using PBMC isolated from a donor carryinghomozygous 158V/V FcγRIII alleles, as described in Example 8.

FIG. 18B. ADCC Assay of Hu1D10-IgG1 and Hu1D10-IgG2M3 Wild-Type andMutant Antibodies Using PBMC From a 158F/F Donor. The ADCC activity ofthe wild-type or mutant Hu1D10-IgG1 and Hu1D10-IgG2M3 antibodies on Rajicells was determined using PBMC isolated from a donor carryinghomozygous 158F/F FcγRIII alleles, as described in Example 8.

FIG. 19. Pharmacokinetics of OST577-IgG2M3 wild-type and variantantibodies in rhesus macaque. The observed and modeled mean serumconcentrations (μg/ml) and standard deviations of OST577-IgG2M3wild-type and variant antibodies administered by infusion at a dose of 1mg/kg to groups of four rhesus macaques were plotted as a function oftime (days after infusion), as described in Example 9.

FIG. 20. Pharmacokinetics of OST577-IgG1 wild-type and variantantibodies in rhesus macaque. The observed and modeled mean serumconcentrations (μg/ml) and standard deviations of OST577-IgG1 wild-typeand variant antibodies administered by infusion at a dose of 1 mg/kg togroups of four rhesus macaques were plotted as a function of time (daysafter infusion), as described in Example 10.

FIG. 21A. pH-Dependent Binding and Release of Hu1D10-IgG3 Wild-Type andMutant Antibodies to Human FcRn. The binding and release of thewild-type or mutant Hu1D10-IgG3 antibodies to human FcRn on transfectedNS0 cells in FBB, at pH 6.0, 6.5, 7.0, 7.5, or 8.0, was analyzed by flowcytometry, as described in Example 11.

FIG. 21B. pH-Dependent Binding and Release of Hu1D10-IgG4 Wild-Type andMutant Antibodies to Human FcRn. The binding and release of thewild-type or mutant Hu1D10-IgG4 antibodies to human FcRn on transfectedNS0 cells in FBB, at pH 6.0, 6.5, 7.0, 7.5, or 8.0, was analyzed by flowcytometry, as described in Example 11.

FIG. 21C. pH-Dependent Binding and Release of Hu1D10-IgG3 Wild-Type andMutant Antibodies to Rhesus FcRn. The binding and release of thewild-type or mutant Hu1D10-IgG3 antibodies to rhesus FcRn on transfectedNS0 cells in FBB, at pH 6.0, 6.5, 7.0, 7.5, or 8.0, was analyzed by flowcytometry, as described in Example 11.

FIG. 21D. pH-Dependent Binding and Release of Hu1D10-IgG4 Wild-Type andMutant Antibodies to Rhesus FcRn. The binding and release of thewild-type or mutant Hu1D10-IgG4 antibodies to rhesus FcRn on transfectedNS0 cells in FBB, at pH 6.0, 6.5, 7.0, 7.5, or 8.0, was analyzed by flowcytometry, as described in Example 11.

FIG. 22. Amino acid sequences of Daclizumab with various FcRn bindingmutations.

FIG. 23. Amino acid sequences of Fontolizumab with various FcRn bindingmutations.

FIG. 24. Amino acid sequences of Visilizumab with various FcRn bindingmutations.

FIG. 25. Amino acid sequences of M200 (volociximab) with various FcRnbinding mutations.

FIG. 26. Pharmacokinetics of Dac-IgG1 wild-type, Dac-IgG1 variant, andDac-IgG2M3 variant antibodies in cynomolgus monkeys. The observed meanserum antibody concentrations (μg/ml) and standard deviations ofDac-IgG1 wild-type, Dac-IgG1 T250Q/M428L, and Dac-IgG2M3 T250Q/M428Lvariant antibodies administered by bolus injection at a dose of 10 mg/kgto groups of ten cynomolgus monkeys were plotted as a function of time(days after injection) along with model predicted curves (simulatedbased on group mean PK parameters), as described in Example 15.

DETAILED DESCRIPTION OF THE INVENTION

I. Modified Antibodies with Altered FcRn Binding Affinity and/or SerumHalf-Lives

In order that the invention may be more completely understood, severaldefinitions are set forth.

As used herein, the terms “immunoglobulin” and “antibody” refer toproteins consisting of one or more polypeptides substantially encoded byimmunoglobulin genes. The recognized immunoglobulin genes include thekappa, lambda, alpha, gamma (γ1, γ2, γ3, γ4), delta, epsilon, and muconstant region genes, as well as the myriad immunoglobulin variableregion genes. Full-length immunoglobulin “light chains” (about 25 Kd or214 amino acids) are encoded by a kappa or lambda variable region geneat the NH2-terminus (about 110 amino acids) and a kappa or lambdaconstant region gene at the COOH-terminus. Full-length immunoglobulin“heavy chains” (about 50 Kd or 446 amino acids) are similarly encoded bya heavy chain variable region gene (about 116 amino acids) and one ofthe other aforementioned constant region genes, e.g., gamma (encodingabout 330 amino acids).

One form of immunoglobulin constitutes the basic structural unit of anantibody. This form is a tetramer and consists of two identical pairs ofimmunoglobulin chains, each pair having one light and one heavy chain.In each pair, the light and heavy chain variable regions are togetherresponsible for binding to an antigen, and the constant regions areresponsible for the antibody effector functions. In addition totetrameric antibodies, immunoglobulins may exist in a variety of otherforms including, for example, Fv, Fab, and (Fab′)₂, as well asbifunctional hybrid antibodies (e.g., Lanzavecchia and Scheidegger, Eur.J. Immunol. 17:105-111 (1987)) and in single chains (e.g., Huston etal., Proc. Natl. Acad. Sci. USA, 85:5879-5883 (1988), and Bird et al.,Science, 242:423-426 (1988), which are incorporated herein byreference). (See, generally, Hood et al., “Immunology”, 2^(nd) ed.,Benjamin, New York (1984), and Hunkapiller and Hood, Nature, 323:15-16(1986), which are incorporated herein by reference).

The term “genetically altered antibodies” refers to antibodies whereinthe sequence of amino acid residues has been changed from that of anative or wild-type antibody. Because of the relevance of recombinantDNA techniques, the present invention is not limited to modification ofamino acid sequences found in natural antibodies. As described below,previously engineered antibodies may be redesigned according to presentinvention in order to obtain the desired characteristics of altered FcRnbinding affinity and/or serum half-life. The possible variants ofmodified antibodies useful with the present invention are many and rangefrom changing just one or a few amino acids to the complete redesign of,for example, the variable or constant region. Changes in the constantregion will, in general, be made in order to improve or altercharacteristics, such as complement fixation, interaction with variousFc-gamma receptors and other effector functions. Changes in the variableregion will be made in order to improve the antigen bindingcharacteristics.

An antibody having a constant region substantially identical to anaturally occurring class IgG antibody constant region refers to anantibody in which any constant region present is substantiallyidentical, i.e., at least about 85-90%, and preferably at least 95%identical, to the amino acid sequence of the naturally occurring classIgG antibody's constant region.

In many preferred uses of the present invention, including in vivo useof the modified antibodies in humans for and in vitro detection assays,it may be preferable to use chimeric, primatized, humanized, or humanantibodies that have been modified (i.e., mutated) according to thepresent invention.

The term “chimeric antibody” refers to an antibody in which the constantregion comes from an antibody of one species (typically human) and thevariable region comes from an antibody of another species (typicallyrodent). Methods for producing chimeric antibodies are known in the art.See e.g., Morrison, Science 229:1202-1207 (1985); Oi et al.,BioTechniques 4:214-221 (1986); Gillies et al., J. Immunol. Methods125:191-202 (1989); U.S. Pat. Nos. 5,807,715; 4,816,567; and 4,816,397,which are incorporated herein by reference in their entireties.

The term “primatized antibody” refers to an antibody comprising monkeyvariable regions and human constant regions. Methods for producingprimatized antibodies are known in the art. See e.g., U.S. Pat. Nos.5,658,570; 5,681,722; and 5,693,780, which are incorporated herein byreference in their entireties.

The term “humanized antibody” or “humanized immunoglobulin” refers to animmunoglobulin comprising a human framework, at least one and preferablyall complementarity determining regions (CDRs) from a non-humanantibody, and in which any constant region present is substantiallyidentical to a human immunoglobulin constant region, i.e., at leastabout 85-90%, and preferably at least 95% identical. Hence, all parts ofa humanized immunoglobulin, except possibly the CDRs, are substantiallyidentical to corresponding parts of one or more native humanimmunoglobulin sequences. Often, framework residues in the humanframework regions will be substituted with the corresponding residuefrom the CDR donor antibody to alter, preferably improve, antigenbinding. These framework substitutions are identified by methods wellknown in the art, e.g., by modeling of the interactions of the CDR andframework residues to identify framework residues important for antigenbinding and sequence comparison to identify unusual framework residuesat particular positions. See, e.g., Queen et al., U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,761; 5,693,762; 6,180,370 (each of which isincorporated by reference in its entirety). Antibodies can be humanizedusing a variety of techniques known in the art including, for example,CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Pat. Nos.5,225,539; 5,530,101 and 5,585,089), veneering or resurfacing (EP592,106; EP 519,596; Padlan, Mol. Immunol., 28:489-498 (1991); Studnickaet al., Prot. Eng. 7:805-814 (1994); Roguska et al., Proc. Natl. Acad.Sci. 91:969-973 (1994), and chain shuffling (U.S. Pat. No. 5,565,332),all of which are hereby incorporated by reference in their entireties.

Completely human antibodies may be desirable for therapeutic treatmentof human patients. Human antibodies can be made by a variety of methodsknown in the art including phage display methods described above usingantibody libraries derived from human immunoglobulin sequences. See U.S.Pat. Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645; WO98/50433; WO 98/24893; WO 98/16654; WO 96/34096; WO 96/33735; and WO91/10741, each of which is incorporated herein by reference in itsentirety.

Human antibodies can also be produced using transgenic mice which areincapable of expressing functional endogenous immunoglobulins, but whichcan express human immunoglobulin genes. For an overview of thistechnology for producing human antibodies, see Lonberg and Huszar, Int.Rev. Immunol. 13:65-93 (1995). For a detailed discussion of thistechnology for producing human antibodies and human monoclonalantibodies and protocols for producing such antibodies, see, e.g., PCTpublications WO 98/24893; WO 92/01047; WO 96/34096; WO 96/33735;European Patent No. 0 598 877; U.S. Pat. Nos. 5,413,923; 5,625,126;5,633,425; 5,569,825; 5,661,016; 5,545,806; 5,814,318; 5,885,793;5,916,771; and 5,939,598, which are incorporated by reference herein intheir entireties. In addition, companies such as Abgenix, Inc. (Fremont,Calif.) and Medarex (Princeton, N.J.) can be engaged to provide humanantibodies directed against a selected antigen using technology similarto that described above.

Completely human antibodies that recognize a selected epitope can begenerated using a technique referred to as “guided selection.” In thisapproach a selected non-human monoclonal antibody, e.g., a mouseantibody, is used to guide the selection of a completely human antibodyrecognizing the same epitope (Jespers et al., Biotechnology 12:899-903(1988).

As used herein the term “altering” may refer to “increasing” or“reducing”.

The present invention provides for a modified antibody of class IgG, inwhich at least one amino acid from the heavy chain constant region,selected from the group consisting of amino acid residues 250, 314, and428, is substituted with an amino acid residue different from thatpresent in the unmodified antibody.

The antibodies of IgG class include antibodies of IgG1, IgG2, IgG3, andIgG4. The constant region of the heavy chain of an IgG molecule isindicated in FIG. 1. The numbering of the residues in the heavy chain isthat of the EU index (Kabat et al., op. cit.). The substitution can bemade at position 250, 314, or 428 alone, or in any combinations thereof,such as at positions 250 and 428, or at positions 250 and 314, or atpositions 314 and 428, or at positions 250, 314, and 428, with positions250 and 428 as a preferred combination.

For each position, the substituting amino acid may be any amino acidresidue different from that present in that position of the unmodifiedantibody.

For position 250, the substituting amino acid residue can be any aminoacid residue other than threonine, including, but not limited to,alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, methionine, asparagine, proline,glutamine, arginine, serine, valine, tryptophan, or tyrosine.

For position 314, the substituting amino acid residue can be any aminoacid residue other than leucine, including, but not limited to, alanine,cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, methionine, asparagine, proline,glutamine, arginine, serine, threonine, valine, tryptophan, or tyrosine.

For position 428, the substituting amino acid residues can be any aminoacid residue other than methionine, including, but not limited to,alanine, cysteine, aspartic acid, glutamic acid, phenylalanine, glycine,histidine, isoleucine, lysine, leucine, asparagine, proline, glutamine,arginine, serine, threonine, valine, tryptophan, or tyrosine. Thepresent invention provides for antibodies of class IgG comprising atleast one of the above-described amino acid substitutions. For example,the present invention provides for the mutated IgG2M3 constant regionscomprising two of the above-mentioned substitutions at position 250,314, and/or 428. The amino acid sequences of some specific substitutions(i.e., mutations) of the constant region provided by the presentinvention are disclosed in Table 1 (SEQ ID NOs: 10-66).

TABLE 1 Substituting Amino Acid 250 314 428 Alanine (A) T250A; SEQ IDNO: 10 L314A; SEQ ID NO: 29 M428A; SEQ ID NO: 48 Cysteine (C) T250C; SEQID NO: 11 L314C; SEQ ID NO: 30 M428C; SEQ ID NO: 49 Aspartic acid (D)T250D; SEQ ID NO: 12 L314D; SEQ ID NO: 31 M428D; SEQ ID NO: 50 Glutamicacid (E) T250E; SEQ ID NO 13 L314E; SEQ ID NO: 32 M428E; SEQ ID NO: 51Phenylalanine (F) T250F; SEQ ID NO: 14 L314F; SEQ ID NO: 33 M428F; SEQID NO: 52 Glycine (G) T250G; SEQ ID NO: 15 L314G; SEQ ID NO: 34 M428G;SEQ ID NO: 53 Histidine (H) T250H; SEQ ID NO: 16 L314H; SEQ ID NO: 35M428H; SEQ ID NO: 54 Isoleucine (I) T250I; SEQ ID NO: 17 L314I; SEQ IDNO: 36 M428I; SEQ ID NO: 55 Lysine (K) T250K; SEQ ID NO: 18 L314K; SEQID NO: 37 M428K; SEQ ID NO: 56 Leucine (L) T250L; SEQ ID NO: 19 WildType M428L; SEQ ID NO: 57 Methionine (M) T250M; SEQ ID NO: 20 L314M; SEQID NO: 38 Wild Type Asparagine (N) T250N; SEQ ID NO: 21 L314N; SEQ IDNO: 39 M428N; SEQ ID NO: 58 Proline (P) T250P; SEQ ID NO: 22 L314P; SEQID NO: 40 M428P; SEQ ID NO: 59 Glutamine (Q) T250Q; SEQ ID NO: 23 L314Q;SEQ ID NO: 41 M428Q; SEQ ID NO: 60 Arginine (R) T250R; SEQ ID NO: 24L314R; SEQ ID NO: 42 M428R; SEQ ID NO: 61 Serine (S) T250S; SEQ ID NO:25 L314S; SEQ ID NO: 43 M428S; SEQ ID NO: 62 Threonine (T) Wild TypeL314T; SEQ ID NO: 44 M428T; SEQ ID NO: 63 Valine (V) T250V; SEQ ID NO:26 L314V; SEQ ID NO: 45 M428V; SEQ ID NO: 64 Tryptophan (W) T250W; SEQID NO: 27 L314W; SEQ ID NO: 46 M428W; SEQ ID NO: 65 Tyrosine (Y) T250Y;SEQ ID NO: 28 L314Y; SEQ ID NO: 47 M428Y; SEQ ID NO: 66

In a preferred embodiment, the present invention provides for a modifiedantibody having an altered serum half-life or FcRn binding affinityrelative to the unmodified antibody. The present invention furtherprovides for a modified antibody of class IgG, in which at least oneamino acid from the heavy chain constant region, selected from the groupconsisting of amino acid residues 250, 314, and 428, is substituted withanother amino acid which is different from that present in theunmodified antibody, thereby altering the binding affinity for FcRnand/or the serum half-life of the modified antibody compared to thebinding affinity and/or serum half-life of said unmodified antibody.

The unmodified antibodies of the present invention include naturalantibodies of all species. The term “natural antibodies” refers to allantibodies produced by a host animal. Non-limiting exemplary naturalantibodies of the present invention include antibodies derived fromhuman, chicken, goats, and rodents (e.g., rats, mice, hamsters andrabbits), including transgenic rodents genetically engineered to producehuman antibodies (see, e.g., Lonberg et al., WO93/12227; U.S. Pat. No.5,545,806; and Kucherlapati et al., WO91/10741; U.S. Pat. No. 6,150,584,which are herein incorporated by reference in their entirety).

The unmodified antibodies of the present invention also includesrecombinant antibodies having the same amino acid sequences as a naturalantibody, or genetically-altered antibodies that have changed amino acidsequences compared to the natural antibodies. They can be made in anyexpression systems including both prokaryotic and eukaryotic expressionsystems or using phage display methods (see, e.g., Dower et al.,WO91/17271 and McCafferty et al., WO92/01047; U.S. Pat. No. 5,969,108,which are herein incorporated by reference in their entirety).

The unmodified antibodies of the present invention also includechimeric, primatized, humanized and human antibodies (see discussionabove). Consequently, a modified antibody of the present invention maybe produced by substituting an amino acid residue at position 250, 314,or 428 in a humanized, primatized, or chimeric antibody, wherein thehumanized, primatized, or chimeric antibody previously has been derivedfrom a native antibody.

Preferably, the chimeric antibodies comprise variable regions derivedfrom rodents and constant regions derived from humans so that thechimeric antibodies have longer half-lives and are less immunogenic whenadministered to a human subject. The primatized antibodies comprisevariable regions derived from primates and constant regions derived fromhumans. The humanized antibodies typically comprise at least one CDRfrom the donor antibodies (for example, murine or chicken antibodies)and heavy and/or light chain human frameworks. Sometimes, some aminoacid residues in the human frameworks will be replaced by the residuesat the equivalent positions of the donor antibodies to ensure the properbinding of the humanized antibodies to their antigens. The detailedguidelines of antibody humanization are disclosed in U.S. Pat. Nos.5,530,101; 5,585,089; 5,693,761; 5,693,762; and 6,180,370, each of whichis incorporated by reference herein.

The unmodified antibodies of the present invention may includegenetically-altered antibodies that are functionally equivalent to thecorresponding natural antibodies. Unmodified antibodies that aregenetically-altered to provide improved stability and/or therapeuticefficacy are preferred. Examples of altered antibodies include thosewith conservative substitutions of amino acid residues, and one or moredeletions or additions of amino acids that do not significantly alterthe antigen binding utility. Substitutions can range from changing ormodifying one or more amino acid residues to complete redesign of aregion as long as the binding or functional utility is maintained.Antibodies of this invention can be altered post-translationally (e.g.,acetylation, and phosphorylation) or can be altered synthetically (e.g.,the attachment of a labeling group).

The unmodified antibodies of the present invention may includeantibodies having enhanced binding affinities for their antigens throughgenetic alterations of the variable regions (see, e.g., U.S. Pat. No.6,350,861, which is herein incorporated by reference in its entirety).In one alternative embodiment, the modified IgGs of the presentinvention that have longer half-lives than wild-type (or unmodified)IgGs may also include IgGs whose bioactive sites, such asantigen-binding sites, Fc-receptor binding sites, or complement-bindingsites, are modified by genetic engineering to increase or reduce suchactivities compared to the wild type.

The unmodified and modified antibodies of the present invention may beof any of the recognized isotypes, but the four IgG isotypes arepreferred, with IgG1 and IgG2 especially preferred. Antibodies withconstant regions mutated to have reduced effector functions, for examplethe IgG2M3 and other IgG2 mutants described in U.S. Pat. No. 5,834,597(which is incorporated by reference in its entirety), are included. In apreferred aspect, the unmodified and modified antibodies in the presentinvention comprise heavy chain constant regions of human IgGs.

The present invention may be applied to any antibody comprising heavychain constant regions of IgG class, preferably IgG1, IgG2, IgG2M3,IgG3, and IgG4. The heavy chain variable regions of such antibodies canbe derived from any selected antibodies. Exemplary antibodies disclosedherein include OST577-IgG1 and OST577-IgG2M3, which comprise the heavychain and light chain variable regions of the human anti-hepatitis Bvirus antibody OST577 (Ehrlich et al., Hum. Antibodies Hybridomas 3:2-7(1992)), the light chain constant region of human lambda-2, and theheavy chain constant region of human IgG1 and IgG2M3, respectively. Alsodisclosed herein are Hu1D10-IgG1 and Hu1D10-IgG2M3, Hu1D10-IgG3 andHu1D10-IgG4 which comprise the heavy and light chain variable regions ofthe humanized anti-HLA-DR β chain allele antibody Hu1D10 (Kostelny etal., Int. J. Cancer 93:556-565 (2001)), the light chain constant regionof human kappa, and the aforementioned heavy chain constant regions ofhuman IgG1, IgG2M3, IgG3 and IgG4, respectively (See, FIG. 3).

Further exemplifications of the present invention disclosed hereininclude mutants of the human IgG1, IgG2M3 (a genetically altered variantof IgG2), IgG3 and IgG4 antibodies, illustrating alteration of the serumhalf-life of an antibody of class IgG. The constant region of the heavychain of IgG2M3 is derived from that of the IgG2 by replacing residues234 and 237 of the IgG2 heavy chain constant region with alanine. Thegeneration of the heavy chain constant region of IgG2M3 is disclosed inU.S. Pat. No. 5,834,597, which is incorporated herein by reference.

Generally, the modified antibodies of the present invention include anyimmunoglobulin molecule that binds (preferably, immunospecifically,i.e., competes off non-specific binding, as determined by immunoassayswell known in the art for assaying specific antigen-antibody binding) anantigen and contains an FcRn-binding fragment. Such antibodies include,but are not limited to, polyclonal, monoclonal, bi-specific,multi-specific, human, humanized, chimeric antibodies, single chainantibodies, Fab fragments, F(ab′)₂ fragments, disulfide-linked Fvs, andfragments containing either a V_(L) or V_(H) domain or even acomplementary determining region (CDR) that specifically binds anantigen, in certain cases, engineered to contain or fused to an FcRnbinding domain.

The modified IgG molecules of the invention may include IgG subclassesof any given animals. For example, in humans, the IgG class includesIgG1, IgG2, IgG3, and IgG4; and the mouse IgG class includes IgG1,IgG2a, IgG2b, and IgG3; and the rat IgG class includes IgG1, IgG2a,IgG2b, IgG2c, and IgG3. It is known that certain IgG subclasses, forexample, rat IgG2b and IgG2c, have higher clearance rates than, forexample, IgG1 (Medesan et al., Eur. J. Immunol., 28:2092-2100 (1998)).Thus, when using IgG subclasses other than IgG lit may be advantageousto substitute one or more of the residues, particularly in the C_(H)2and C_(H)3 domains, which differ from the IgG1 sequence with those ofIgG1, thereby increasing the in vivo half-life of the other types ofIgG.

The immunoglobulins of the present invention may be from any animalorigin including birds and mammals. Preferably, the antibodies arehuman, rodent, donkey, sheep, rabbit, goat, guinea pig, camel, horse, orchicken. As used herein, “human” antibodies include antibodies havingthe amino acid sequence of a human immunoglobulin and include antibodiesisolated from human immunoglobulin libraries or from animals transgenicfor one or more human immunoglobulin, as described infra and, forexample, in U.S. Pat. No. 5,939,598 by Kucherlapati et al.

In addition, the modified antibodies of the present invention may bemonospecific, bispecific, trispecific antibodies or of greatermultispecificity. Multispecific antibodies may be specific for differentepitopes of a polypeptide or may be specific for heterologous epitopes,such as a heterologous polypeptide or solid support material. See, e.g.,PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793;Tutt et al., J. Immunol. 147:60-69 (1991); U.S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; Kostelny et al., J. Immunol.148:1547-1553 (1992).

The modified antibodies of the invention include derivatives that areotherwise modified, i.e., by the covalent attachment of any type ofmolecule to the antibody such that covalent attachment does not preventthe antibody from binding antigen and/or generating an anti-idiotypicresponse. For example, but not by way of limitation, the antibodyderivatives include antibodies that have been modified, e.g., byglycosylation, acetylation, pegylation, phosphorylation, amidation,derivatization by known protecting/blocking groups, proteolyticcleavage, linkage to a cellular ligand or other protein, etc. Any ofnumerous chemical modifications may be carried out by known techniques,including, but not limited to, specific chemical cleavage, acetylation,formylation, metabolic synthesis of tunicamycin, etc. Additionally, thederivative may contain one or more non-classical amino acids.

Monoclonal antibodies useful with the present invention can be preparedusing a wide variety of techniques known in the art including the use ofhybridoma, recombinant, and phage display technologies, or a combinationthereof. For example, monoclonal antibodies can be produced usinghybridoma techniques including those known in the art and taught, forexample, in Harlow and Lane, “Antibodies: A Laboratory Manual,” ColdSpring Harbor Laboratory Press, New York (1988); Hammerling et al., in:“Monoclonal Antibodies and T-Cell Hybridomas,” Elsevier, New York(1981), pp. 563-681 (both of which are incorporated herein by referencein their entireties).

The term “monoclonal antibody” as used herein is not limited toantibodies produced through hybridoma technology. The term “monoclonalantibody” refers to an antibody that is derived from a single clone,including any eukaryotic, prokaryotic, or phage clone, and not themethod by which it is produced.

Methods for producing and screening for specific antibodies usinghybridoma technology are routine and well known in the art. In anon-limiting example, mice can be immunized with an antigen of interestor a cell expressing such an antigen. Once an immune response isdetected, e.g., antibodies specific for the antigen are detected in themouse serum, the mouse spleen is harvested and splenocytes isolated. Thesplenocytes are then fused by well-known techniques to any suitablemyeloma cells. Hybridomas are selected and cloned by limiting dilution.The hybridoma clones are then assayed by methods known in the art forcells that secrete antibodies capable of binding the antigen. Ascitesfluid, which generally contains high levels of antibodies, can begenerated by inoculating mice intraperitoneally with positive hybridomaclones.

Antibody fragments that recognize specific epitopes may also be usefulwith the present invention and may be generated by well-knowntechniques. For example, Fab and F(ab′)₂ fragments may be produced byproteolytic cleavage of immunoglobulin molecules, using enzymes such aspapain (to produce Fab fragments) or pepsin (to produce F(ab′)₂fragments). F(ab′)₂ fragments contain the complete light chain, and thevariable region, the CH1 region and the hinge region of the heavy chain.

For example, antibodies can also be generated using various displaymethods known in the art, including phage display. In phage displaymethods, functional antibody domains are displayed on the surface ofphage particles that carry the polynucleotide sequences encoding them.In a particular embodiment, such phage can be utilized to displayantigen-binding domains, such as Fab and Fv or disulfide-bond stabilizedFv, expressed from a repertoire or combinatorial antibody library (e.g.,human or murine). Phage expressing an antigen binding domain that bindsthe antigen of interest can be selected or identified with antigen,e.g., using labeled antigen or antigen bound or captured to a solidsurface or bead. Phage used in these methods are typically filamentousphage, including fd and M13. The antigen binding domains are expressedas a recombinantly fused protein to either the phage gene III or geneVIII protein. Alternatively, the modified FcRn binding portion ofimmunoglobulins of the present invention can be also expressed in aphage display system. Examples of phage display methods that can be usedto make the immunoglobulins, or fragments thereof, of the presentinvention include those disclosed in Brinkman et al., J. Immunol.Methods 182:41-50 (1995); Ames et al., J. Immunol. Methods 184:177-186(1995); Kettleborough et al., Eur. J. Immunol. 24:952-958 (1994); Persicet al., Gene 187:9-18 (1997); Burton et al., Advances in Immunology57:191-280 (1994); PCT application No. PCT/GB91/01134; PCT publicationsWO 90/02809; WO 91/10737; WO 92/01047; WO 92/18619; WO 93/11236; WO95/15982; WO 95/20401; and U.S. Pat. Nos. 5,698,426; 5,223,409;5,403,484; 5,580,717; 5,427,908; 5,750,753; 5,821,047; 5,571,698;5,427,908; 5,516,637; 5,780,225; 5,658,727; 5,733,743 and 5,969,108;each of which is incorporated herein by reference in its entirety.

As described in the above references, after phage selection, theantibody coding regions from the phage can be isolated and used togenerate whole antibodies, including human antibodies, or any otherdesired fragments, and expressed in any desired host, includingmammalian cells, insect cells, plant cells, yeast, and bacteria, e.g.,as described in detail below. For example, techniques to recombinantlyproduce Fab, Fab′, and F(ab′)₂ fragments can also be employed usingmethods known in the art such as those disclosed in PCT publication WO92/22324; Mullinax et al., BioTechniques 12:864-869 (1992); Sawai etal., Amer. J. Reprod. Immunol. 34:26-34 (1995); and Better et al.,Science 240:1041-1043 (1988), each of which is incorporated by referencein its entirety. Examples of techniques which can be used to producesingle-chain Fvs and antibodies include those described in U.S. Pat.Nos. 4,946,778 and 5,258,498; Huston et al., Methods in Enzymology203:46-88 (1991); Shu et al., Proc. Natl. Acad. Sci. 90:7995-7999(1993); and Skerra et al., Science 240:1038-1040 (1988).

In particular embodiments, the modified antibodies have in vivotherapeutic and/or prophylactic uses. Examples of therapeutic andprophylactic antibodies which may be so modified include, but are notlimited to, SYNAGIS® (palivizumab) (Medimmune, MD) which is a humanizedanti-respiratory syncytial virus (RSV) monoclonal antibody for thetreatment of patients with RSV infection; HERCEPTIN® (trastuzumab)(Genentech, CA) which is a humanized anti-HER2 monoclonal antibody forthe treatment of patients with metastatic breast cancer; REMICADE®(infliximab) (Centocor, Pa.) which is a chimeric anti-TNF-α monoclonalantibody for the treatment of patients with Crohn's disease; REOPRO®(abciximab) (Centocor) which is an anti-glycoprotein IIb/IIIa receptoron the platelets for the prevention of clot formation; ZENAPAX®(daclizumab) (Roche Pharmaceuticals, Switzerland) which is animmunosuppressive, humanized anti-CD25 monoclonal antibody for theprevention of acute renal allograft rejection. Other examples are ahumanized anti-CD18 F(ab′)₂ (Genentech); CDP860 which is a humanizedanti-CD18 F (ab′)₂ (Celltech, UK); PR0542 which is an anti-HIV gp120antibody fused with CD4 (Progenics/Genzyme Transgenics); OSTAVIR™ whichis a human anti Hepatitis B virus antibody (Protein DesignLabs/Novartis); PROTOVIR™ which is a humanized anti-CMV IgG1 antibody(Protein Design Labs/Novartis); IC14 which is an anti-CD14 antibody(ICOS); AVASTIN™ which is a humanized anti-VEGF IgG1 antibody(Genentech); ERBITUX™ which is a chimeric anti-EGFR IgG antibody(ImClone Systems); VITAXIN™ which is a humanized anti-αVβ3 integrinantibody (Applied Molecular Evolution/Medimmune); Campath-1H/LDP-03which is a humanized anti-CD52 IgG1 antibody (Leukosite); ZAMYL™ whichis a humanized anti-CD33 IgG antibody (Protein Design Labs/Kanebo);RITUXAN™ which is a chimeric anti-CD20 IgG1 antibody (IDECPharmaceuticals/Genentech, Roche/Zenyaku); LYMPHOCIDE™ which is ahumanized anti-CD22 IgG antibody (Immunomedics); REMITOGEN™ which is ahumanized anti-HLA-DR antibody (Protein Design Labs); ABX-IL8 which is ahuman anti-IL8 antibody (Abgenix); RAPTIVA™ which is a humanized IgG1antibody (Genetech/Xoma); ICM3 which is a humanized anti-ICAM3 antibody(ICOS); IDEC-114 which is a primatized anti-CD80 antibody (IDECPharmaceuticals/Mitsubishi); IDEC-131 which is a humanized anti-CD40Lantibody (IDEC/Eisai); IDEC-151 which is a primatized anti-CD4 antibody(IDEC); IDEC-152 which is a primatized anti-CD23 antibody(IDEC/Seikagaku); NUVION™ which is a humanized anti-CD3 IgG2M3 antibody(Protein Design Labs); 5G1.1 which is a humanized anti-complement factor5 (C5) antibody (Alexion Pharmaceuticals); HUMIRA™ which is a humananti-TNF-α antibody (CAT/BASF); CDP870 which is a humanized anti-TNF-αFab fragment (Celltech); IDEC-151 which is a primatized anti-CD4 IgG1antibody (IDEC Pharmaceuticals/Smith-Kline Beecham); MDX-CD4 which is ahuman anti-CD4 IgG antibody (Medarex/Eisai/Genmab); CDP571 which is ahumanized anti-TNF-α IgG4 antibody (Celltech); LDP-02 which is ahumanized anti-α4β7 antibody (LeukoSite/Genentech); OrthoClone OKT4Awhich is a humanized anti-CD4 IgG antibody (Ortho Biotech); ANTOVA™which is a humanized anti-CD40L IgG antibody (Biogen); TYSABRI® which isa humanized anti-VLA-4 IgG antibody (Elan); MDX-33 which is a humananti-CD64 (FcγR) antibody (Medarex/Centeon); SCH55700 which is ahumanized anti-IL-5 IgG4 antibody (Celltech/Schering); SB-240563 andSB-240683 which are humanized anti-IL-5 and IL-4 antibodies,respectively (SmithKline Beecham); rhuMab-E25 which is a humanizedanti-IgE IgG1 antibody (Genentech/Novartis/Tanox Biosystems); IDEC-152which is a primatized anti-CD23 antibody (IDEC Pharmaceuticals);SIMULECT™ which is a chimeric anti-CD25 IgG1 antibody (NovartisPharmaceuticals); LDP-01 which is a humanized anti-β2-integrin IgGantibody (Leukosite); CAT-152 which is a human anti-TGF-β2 antibody(Cambridge Antibody Technology); and Corsevin M which is a chimericanti-Factor VII antibody (Centocor).

The present invention permits modification of these and othertherapeutic antibodies to increase the in vivo half-life, allowingadministration of lower effective dosages and/or less frequent dosing ofthe therapeutic antibody. Such modification to increase in vivohalf-life can also be useful to improve diagnostic immunoglobulins aswell. For example, increased serum half-life of a diagnostic antibodymay permit administration of lower doses to achieve sufficientdiagnostic sensitivity. Alternatively, decreased serum half-life may beadvantageous in applications where rapid clearance of a diagnosticantibody is desired.

Disclosed herein is the amino acid sequence of OST577-IgG2M3, includingthe amino acid sequence of its heavy chain variable region (SEQ IDNO: 1) (OST577-VH) and constant region (SEQ ID NO: 2) (IgG2M3-CH) withpositions 250, 314, and 428 highlighted, and the amino acid sequence ofits light chain variable region (SEQ ID NO: 4) (OST577-VL) and constantregion (SEQ ID NO: 5) (LAMBDA2-CL).

Disclosed herein is the amino acid sequence of the heavy chain constantregion of OST577-IgG1 (SEQ ID NO: 3).

The present invention provides for a modified antibody having anincreased binding affinity for FcRn and/or an increased serum half-lifeas compared with the unmodified antibody, wherein amino acid residue 250or 428 from the heavy chain constant region is substituted with anotheramino acid residue that is different from that present in the unmodifiedantibody. Preferably, amino acid residue 250 from the heavy chainconstant region is substituted with glutamic acid or glutamine.Alternatively, amino acid residue 428 from the heavy chain constantregion is substituted with phenylalanine or leucine.

In one example, said unmodified antibody comprises the heavy chainconstant region of an IgG1, or IgG2, or IgG2M3 molecule, including, butnot limited to, OST577-IgG2M3 or OST577-IgG1. IgG1, IgG2, and IgG2M3have a threonine residue at position 250 and a methionine residue atposition 428. Preferably, the threonine residue at position 250 issubstituted with glutamic acid (T250E) or glutamine (T250Q), and themethionine residue at position 428 is substituted with phenylalanine(M428F) or leucine (M428L). The amino acid sequences of the heavy chainconstant region of the modified IgG2M3 having the amino acidsubstitutions of T250E (SEQ ID NO: 13), T250Q (SEQ ID NO: 23), M428F(SEQ ID NO: 52), or M428L (SEQ ID NO: 57) are disclosed herein. Theamino acid sequences of the constant region of the heavy chain of themodified IgG1 having the amino acid substitutions of T250D (SEQ ID NO:67), T250E (SEQ ID NO: 68), T250Q (SEQ ID NO: 69), M428F (SEQ ID NO:70), or M428L (SEQ ID NO. 71) are also disclosed herein.

The present invention provides for a modified antibody having anincreased binding affinity for FcRn and/or an increased serum half-lifeas compared with the unmodified antibody. The amino acid modificationcan be any one of the following substitutions:

-   -   1) Amino acid residue 250 from the heavy chain constant region        is substituted with glutamic acid and amino acid residue 428        from the heavy chain constant region is substituted with        phenylalanine.    -   2) Amino acid residue 250 from the heavy chain constant region        is substituted with glutamine and amino acid residue 428 from        the heavy chain constant region is substituted with        phenylalanine.    -   3) Amino acid residue 250 from the heavy chain constant region        is substituted with glutamine and amino acid residue 428 from        the heavy chain constant region is substituted with leucine.

The amino acid sequences of the heavy chain constant region of themodified IgG2M3 having the double amino acid substitutions ofT250E/M428F (SEQ ID NO: 72), T250Q/M428F (SEQ ID NO: 73), or T250Q/M428L(SEQ ID NO: 74) are disclosed herein.

The amino acid sequences of the heavy chain constant region of themodified IgG1 having the double amino acid substitutions of T250E/M428F(SEQ ID NO: 75) or T250Q/M428L (SEQ ID NO: 76) are disclosed herein.

The modified antibodies with the described double amino acidsubstitutions at positions 250 and 428 display exceedingly high bindingaffinities for FcRn compared to that of the unmodified antibodies.

In a preferred embodiment of the present invention, the binding affinityfor FcRn and/or the serum half-life of the modified antibody isincreased by at least about 30%, 50%, 80%, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 40-fold, 50-fold,60-fold, 70-fold, 80-fold, 90-fold, or 100-fold.

The present invention provides for a modified antibody having a reducedbinding affinity for FcRn and/or a reduced serum half-life as comparedwith the unmodified antibody, wherein amino acid residue 314 from theheavy chain constant region is substituted with another amino acid whichis different from that present in an unmodified antibody. The modifiedantibodies having an amino acid substitution at position 314 have beenshown to display a reduced binding affinity, suggesting that position314 should be modified if a reduced serum half-life of an antibody isdesired. Preferably, the amino acid residue 314 from the heavy chainconstant region is substituted with alanine, arginine, aspartic acid,asparagine, cysteine, glutamic acid, glutamine, glycine, histidine,lysine, methionine, phenylalanine, proline, serine, threonine,tryptophan, tyrosine, or valine. More preferably, the amino acidsubstitution is from leucine to alanine or arginine at position 314. Asshown in the Examples, the binding affinity for FcRn of the modifiedOST577-IgG2M3 comprising a substitution from leucine to arginine isreduced to 11% of that of the unmodified OST577-IgG2M3.

The amino acid sequence of the heavy chain constant region of themodified IgG2M3, having the amino acid substitution from leucine toalanine at position 314 is disclosed herein as SEQ ID NO: 29. The aminoacid sequence of the heavy chain constant region of the modified IgG2M3,having the amino acid substitution from leucine to arginine at position314 is disclosed herein as SEQ ID NO: 42.

The present invention provides for a modified antibody having a reducedbinding affinity for FcRn and/or a reduced serum half-life as comparedwith the unmodified antibody, wherein (1) amino acid residue 250 fromthe heavy chain constant region is substituted with arginine,asparagine, aspartic acid, lysine, phenylalanine, proline, tryptophan,or tyrosine; or (2) amino acid residue 428 from the heavy chain constantregion is substituted with alanine, arginine, asparagine, aspartic acid,cysteine, glutamic acid, glutamine, glycine, histidine, lysine, proline,serine, threonine, tyrosine, or valine. Preferably, amino acid residue250 from the heavy chain constant region is substituted with asparticacid, or amino acid residue 428 from the heavy chain constant region issubstituted with glycine. Such an amino acid substitution candramatically reduce the serum half-life of an antibody. As shown in theExamples, the binding affinity for FcRn of the modified OST577-IgG2M3having such amino acid substitutions is reduced to about 5-7% of that ofthe unmodified OST577-IgG2M3.

The amino acid sequence of the heavy chain constant region of themodified IgG2M3 having the amino acid substitution from threonine toaspartic acid at position 250 is disclosed herein as SEQ ID NO: 12. Theamino acid sequence of the heavy chain constant region of the modifiedIgG2M3 having the amino acid substitution from methionine to glycine atposition 428 is disclosed herein as SEQ ID NO: 53.

In a preferred embodiment of the present invention, the binding affinityfor FcRn and/or the serum half-life of said modified antibody is reducedby at least about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%,98%, or 99%.

The present invention includes the heavy chain constant regions, Fcregions, or C_(H)2-C_(H)3 regions of the modified IgG antibodiesdescribed herein, preferably, of the modified IgG1, IgG2 or IgG2M3antibodies having the amino acid substitutions described herein.

The present invention also includes a polypeptide comprising an aminoacid sequence of any one of the SEQ ID Nos: 10-76. In a preferredembodiment, these polypeptides are mutated IgG1, IgG2, or IgG2M3constant regions.

The heavy chain constant regions of the modified antibodies of thepresent invention can be linked to the heavy chain variable region ofany selected antibody to generate the desired hybrid heavy chain.Examples of the selected antibodies include, but are not limited to,antibodies against IL-2, IL-4, IL-10, IL-12, HSV, CD3, CD33, CMV, andIFN-γ. In addition, the variable regions can be those of naturalantibodies of any species such as a human, a primate, or a rodent.Alternatively, they can be that of genetically-altered antibodies,including, but not limited to, humanized antibodies, antibodies havingincreased binding affinities to their antigen through geneticmodification, or fully human antibodies. Such a hybrid heavy chain canbe linked to a variety of light chains to produce the desired antibody.The light chains can be either lambda or kappa light chains. Since theserum half-life of an antibody is determined primarily by its heavychain constant region, a desired serum half-life of a produced antibodycan be accomplished through the amino acid substitutions in the heavychain constant region described herein.

II. Production of Modified Antibodies with Altered FcRn Binding Affinityand/or Serum Half-lives

The present invention provides for methods of producing proteins,particularly antibodies with altered FcRn binding affinity and/or serumhalf-lives. Preferably, the present invention provides for methods tomodify a given antibody of class IgG at one or more of the positionsdisclosed herein. This may be achieved chemically, or by random orsite-directed mutagenesis and recombinant production using any knownproduction methods.

The present invention provides for a method of modifying an antibody ofclass IgG, comprising substituting at least one amino acid from theheavy chain constant region selected from the group consisting of aminoacid residues 250, 314, and 428 with an amino acid which is differentfrom that present in an unmodified antibody, thereby causing alterationof the binding affinity for FcRn and/or the serum half-life of saidunmodified antibody.

The substitution can be made at position 250, 314, or 428 alone, or inany combinations thereof, such as at positions 250 and 428.

To increase the binding affinity for FcRn and/or increase the serumhalf-life of an antibody, amino acid residue 250 from the heavy chainconstant region is substituted with glutamic acid or glutamine, or aminoacid residue 428 from the heavy chain constant region is substitutedwith phenylalanine or leucine. Alternatively, amino acid residue 250from the heavy chain constant region is substituted with glutamic acidand amino acid residue 428 from the heavy chain constant region issubstituted with phenylalanine; or amino acid residue 250 from the heavychain constant region is substituted with glutamine and amino acidresidue 428 from the heavy chain constant region is substituted withphenylalanine; or amino acid residue 250 from the heavy chain constantregion is substituted with glutamine and amino acid residue 428 from theheavy chain constant region is substituted with leucine. Modification atboth 250 and 428 is preferred since antibodies having such doublemutations display exceptionally high binding affinities for FcRn.

To produce a modified antibody having a reduced binding affinity forFcRn and/or reduced serum half-life, as compared with the unmodifiedantibody, amino acid residue 314 from the heavy chain constant region issubstituted with another amino acid, which is different from thatpresent in an unmodified antibody. Preferably, amino acid residue 314from the heavy chain constant region is substituted with alanine,arginine, aspartic acid, asparagine, cysteine, glutamic acid, glutamine,glycine, histidine, lysine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, or valine. More preferably, amino acidresidue 314 from the heavy chain constant region is substituted withalanine or arginine.

To produce a modified antibody having a reduced binding affinity forFcRn and a reduced serum half-life as compared with the unmodifiedantibody, amino acid residue 250 from the heavy chain constant region issubstituted with arginine, asparagine, aspartic acid, lysine,phenylalanine, proline, tryptophan, or tyrosine; or amino acid residue428 from the heavy chain constant region is substituted with alanine,arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine,glycine, histidine, lysine, proline, serine, threonine, tyrosine, orvaline. More preferably, amino acid residue 250 is substituted withaspartic acid or amino acid 428 is substituted with glycine.

The amino acid substitutions described herein are achieved by standardrecombinant DNA technology. In one embodiment, site-directed mutagenesismay be used to introduce the amino acid substitutions into the DNAencoding an unmodified antibody. The resulting DNAs of modifiedantibodies are then delivered into host cells and the modifiedantibodies are so produced. The desired alteration of binding affinityfor FcRn of the modified antibodies can be selected by using phagedisplay technology or any other suitable methods known in the art andconfirmed by measuring the binding affinity.

Preferably, a method of producing modified antibody of class IgG with analtered binding affinity for FcRn and an altered serum half-life ascompared with unmodified antibody comprises:

(a) preparing a replicable expression vector comprising a suitablepromoter operably linked to a DNA which encodes at least a constantregion of an immunoglobulin heavy chain and in which at least one aminoacid from the heavy chain constant region selected from the groupconsisting of amino acid residues 250, 314, and 428 is substituted withan amino acid which is different from that present in an unmodifiedantibody thereby causing an alteration in serum half-life;

(b) transforming host cells with said vector; and

(c) culturing said transformed host cells to produce said modifiedantibody.

Such a method optionally further comprises after Step (a): preparing asecond replicable expression vector comprising a promoter operablylinked to a DNA which encodes a complementary immunoglobulin light chainand wherein said cell line is further transformed with said vector.

To generate the DNA in Step (a), the amino acid substitutions can beintroduced by mutagenesis, including, but not limited to, site-directedmutagenesis (Kunkel, Proc. Natl. Acad. Sci. USA 82:488-492 (1985)), PCRmutagenesis (Higuchi, in “PCR Protocols: A Guide to Methods andApplications”, Academic Press, San Diego (1990) pp. 177-183), andcassette mutagenesis (Wells et al., Gene 34:315-323 (1985)). Preferably,site-directed mutagenesis is performed by the overlap-extension PCRmethod, which is disclosed in the Examples (Higuchi, in “PCR Technology:Principles and Applications for DNA Amplification”, Stockton Press, NewYork (1989), pp. 61-70).

The technique of overlap-extension PCR (Higuchi, ibid.) can be used tointroduce any desired mutation(s) into a target sequence (the startingDNA). For example, as shown in FIG. 4, the first round of PCR in theoverlap-extension method involves amplifying the target sequence with anoutside primer (primer 1) and an internal mutagenesis primer (primer 3),and separately with a second outside primer (primer 4) and an internalprimer (primer 2), yielding two PCR segments (segments A and B). Theinternal mutagenesis primer (primer 3) is designed to contain mismatchesto the target sequence specifying the desired mutation(s). In the secondround of PCR, the products of the first round of PCR (segments A and B)are amplified by PCR using the two outside primers (primers 1 and 4).The resulting full-length PCR segment (segment C) is digested withrestriction enzymes and the resulting restriction fragment is clonedinto an appropriate vector.

As the first step of mutagenesis, the starting DNA is operably clonedinto a mutagenesis vector. The primers are designed to reflect thedesired amino acid substitution (see more details in the Examples). Inone example, the vectors used for in vitro mutagenesis can be used fordirecting protein expression. Thus, the resulting DNA of theoverlap-extension PCR can be cloned back into the mutagenesis vector sothat an expression vector comprising the DNA with the desired mutationis created. An example of the mutagenesis vector comprising the startingDNA includes, but is not limited to, pVAg2M3-OST577.

For example, the mutations at position 250 were made by amplifying theregion surrounding the PinAI-BamHI fragment of pVAg2M3-OST577 (see therestriction map in FIG. 5A) in a two step process using theoverlap-extension method described above, then digesting the resultingPCR segment with PinAI and BamHI, and cloning the resulting restrictionfragment into pVAg2M3-OST577. Similarly, the mutations at position 314or 428 were made by amplifying the region surrounding the PmlI-BamHIfragment by overlap-extension PCR, then digesting the resulting PCRsegments with PmlI and BamHI, and cloning the resulting restrictionfragments into pVAg2M3-OST577.

The starting DNA can be a DNA encoding an entire unmodified antibody, anentire immunoglobulin heavy chain of an unmodified antibody, theconstant region of a heavy chain, or part of the heavy chain constantregion of an unmodified antibody as long as the amino acid residue thatis going to be modified is included.

If the DNA encoding an entire unmodified antibody is used as thestarting DNA for mutagenesis, the entire modified antibody can beproduced by performing Steps (a), (b), and (c) of the method describedherein. The step between Step (a) and Step (b) of said method forgenerating the complementary light chain would not be necessary.

If the starting DNA for mutagenesis is a DNA encoding the entire heavychain of an unmodified antibody, mutagenesis will give rise to a vectorcomprising the DNA encoding the entire modified heavy chain. In order toproduce an entire modified antibody, the step between Steps (a) and (b)of the method disclosed herein is performed. That is, another replicableexpression vector comprising a suitable promoter operably linked to aDNA encoding the complementary immunoglobulin light chain isco-transfected into the same host cells. As a result, both thecomplementary light chain and the modified heavy chain are expressed inthe same host cells and assembled properly to give rise to the entiremodified antibody. An example of said expression vector comprising a DNAencoding an immunoglobulin light chain includes, but is not limited to,pVAλ2-OST577.

If the starting DNA for mutagenesis is a DNA encoding part of the heavychain constant region, such as a C_(H)2-C_(H)3 segment or an Fc domain,the resulting DNA encoding such a modified partial heavy chain is firstconnected in frame with the remaining unmodified heavy chain, so thatthe DNA encoding an entire heavy chain with the modification describedherein in Step (a) is generated. An entire modified antibody is thenproduced by co-transfecting host cells with the vector comprising theDNA encoding a complementary light chain and the vector comprising theDNA encoding such modified heavy chain. The connection of the DNAencoding the modified partial heavy chain and the remaining unmodifiedheavy chain can be achieved by using the standard molecular cloningtechniques known in the art of molecular biology, such as restrictiondigestions and ligations (Sambrook and Russell, “Molecular Cloning: ALaboratory Manual”, 3^(rd) edition, Cold Spring Harbor Laboratory Press,New York (2001)).

The light and heavy chains can be cloned in the same or differentexpression vectors. The DNA segments encoding immunoglobulin chains areoperably linked to control sequences in the expression vector(s) thatensure the expression of immunoglobulin polypeptides. Such controlsequences include a signal sequence, a promoter, an enhancer, and atranscription termination sequence (see Queen et al., Proc. Natl. Acad.Sci. USA 86:10029-10033 (1989); WO90/07861; Co et al., J. Immunol.148:1149-1154 (1992); “Antibody Engineering: A Practical Guide”,Borrebaeck, Ed., Freeman, New York (1997)) which are incorporated hereinby reference in their entirety for all purposes).

Host cells are transformed by using the techniques known in the art,such as liposome, calcium phosphate, electroporation, etc. (Sambrook andRussell, op. cit.). Preferably, the host cells are transientlytransfected using the liposome method.

The host cells used to produce the modified antibody of the presentinvention may be cultured in a variety of media known in the arts.

Modified antibodies described herein can be produced intracellularly, inthe periplasmic space, or directly secreted into the medium. Preferably,the modified antibodies in the present invention are secreted intoculture media. The media of the host cell culture producing modifiedantibodies are collected and cell debris is spun down by centrifugation.The supernatants are collected and subjected to the protein expressionassays (see more details in the Examples).

The expression of a modified antibody is confirmed by gelelectrophoresis using SDS-PAGE reducing or non-reducing protein gelanalysis, or any other techniques known in the art. ELISA can also beused to detect both the expression of a modified antibody and thequantity of that antibody.

Modified antibodies should retain proper binding to antigens as comparedwith the unmodified antibodies. Accordingly, proper antibody-antigenbinding is tested by the procedures known in the art of immunology, suchas ELISA. Additional experiments can be performed to confirm thatmodified antibodies have structural properties similar to those ofunmodified antibodies. These experiments include, but are not limitedto, SDS-PAGE, SEC, ELISA, and protein A binding assays. The protein Abinding assay is preferred since protein A binds to the same region ofthe C_(H)2-C_(H)3 junction as FcRn, although the binding involvesdifferent residues.

Modified antibodies prepared from host cells can be purified using thetechniques known in the art, including, but not limited to, gelfiltration and column chromatography (e.g., affinity chromatography byprotein A, cation exchange chromatography, anion exchangechromatography, and gel filtration). The minimum acceptable purity ofthe antibody for use in pharmaceutical formulation will be 90%, with 95%preferred, 98% more preferred and 99% or higher the most preferred.

The binding affinities of the produced antibodies for FcRn can bedetected by performing a competitive binding assay at pH 6.0, theoptimal condition for binding to FcRn. The binding affinities can betested by immobilizing FcRn on a solid substrate such as a Sepharose®bead. Alternatively, the binding affinities can be evaluated using anELISA. Preferably, the present invention tests the binding affinities bycarrying out a competitive binding assay in a cell-based system. Adilution series of a produced modified antibody and the unmodifiedantibody are compared for binding to FcRn expressed on a cell line,preferably an NS0 cell line. The experimental procedures for carryingout a competitive binding assay are described in detail in the Examples.

The experiments in the present invention show that similar bindingaffinity results can be achieved with purified antibodies or culturesupernatants of the cells producing antibodies. Accordingly,supernatants can be used directly to test the binding affinities forFcRn of the produced antibodies in order to confirm that the desiredalteration of the binding affinities has been accomplished. After such aconfirmation, the produced antibody is subjected to more complexpurification procedures.

Direct binding assays should also be performed to confirm that themodified antibodies bind to the FcRn in a pH-dependent manner. Inparticular, the binding affinity of the modified antibodies for FcRn istested both at pH 6.0 and at pH 8.0 (see more details in Examples). Ingeneral, the binding affinity at pH 6.0 should exceed that at pH 8.0.

Biological stability (or serum half-life) may be measured by a varietyof in vitro or in vivo means, for example, by using a radiolabeledprotein and measuring levels of serum radioactivity as a function oftime, or by assaying the levels of intact antibody (of knownspecificity) present in the serum using ELISA as a function of time,with a particularly preferred measure of increased biological stabilitybeing evidenced by increased serum half-life and decreased clearancerates.

The present invention provides for the polynucleotide molecules encodingthe modified antibodies, or the polynucleotide molecules encoding amodified partial or full heavy chain of the modified antibodies, such asthe constant regions, Fc regions or C_(H)2-C_(H)3 regions, with themutations described herein.

The present invention provides for the vectors comprising thepolynucleotide molecules encoding the modified antibodies, or thepolynucleotide molecules encoding the modified partial or full heavychains of the modified antibodies, such as the constant regions, Fcregions or C_(H)2-C_(H)3 regions, with the mutations (substitutions)described herein.

The present invention includes a host cell containing said vectorscomprising said nucleic acid molecules as described herein. Suitablehost cells for the expression of the modified antibodies describedherein are derived from prokaryotic organisms such as Escherichia coli,or eukaryotic multi-cellular organisms, including yeasts, plants,insects, and mammals.

E. coli is one prokaryotic host particularly useful for cloning and/orexpressing the DNA sequences of the present invention. Other microbialhosts suitable for use include bacilli, such as Bacillus subtilis, andother enterobacteriaceae, such as Salmonella, Serratia, and variousPseudomonas species. In these prokaryotic hosts, one can also makeexpression vectors, which typically contain expression control sequencescompatible with the host cell (e.g., an origin of replication). Inaddition, any number of a variety of well-known promoters can bepresent, such as the lactose promoter system, a tryptophan (trp)promoter system, a beta-lactamase promoter system, or a promoter systemfrom phage lambda. The promoters typically control expression,optionally with an operator sequence, and have ribosome binding sitesequences and the like, for initiating and completing transcription andtranslation.

Other microbes, such as yeast, can also be used for expression.Saccharomyces is a preferred host, with suitable vectors havingexpression control sequences, such as promoters, including3-phosphoglycerate kinase or other glycolytic enzymes, and an origin ofreplication, termination sequences and the like as desired.

Plants and plant cell cultures can be used for expression of the DNAsequence of the invention (Larrick and Fry, Hum. Antibodies Hybridomas2:172-189 (1991); Benvenuto et al., Plant Mol. Biol. 17:865-874 (1991);During et al., Plant Mol. Biol. 15:281-293 (1990); Hiatt et al., Nature342:76-78 (1989)). Preferable plant hosts include, for example:Arabidopsis, Nicotiana tabacum, Nicotiana rustica, and Solanumtuberosum. A preferred expression cassette for expressing polynucleotidesequences encoding the modified antibodies of the invention is theplasmid pMOG18 in which the inserted polynucleotide sequence encodingthe modified antibody is operably linked to a CaMV 35S promoter with aduplicated enhancer; pMOG18 is used according to the method of Sijmonset al., Bio/Technology 8:217-221 (1990). Alternatively, a preferredembodiment for the expression of modified antibodies in plants followsthe methods of Hiatt et al., supra, with the substitution ofpolynucleotide sequences encoding the modified antibodies of theinvention for the immunoglobulin sequences used by Hiatt et al., supra.Agrobacterium tumifaciens T-DNA-based vectors can also be used forexpressing the DNA sequences of the invention; preferably such vectorsinclude a marker gene encoding spectinomycin-resistance or anotherselectable marker.

Insect cell culture can also be used to produce the modified antibodiesof the invention, typically using a baculovirus-based expression system.The modified antibodies can be produced by expressing polynucleotidesequences encoding the modified antibodies according to the methods ofPutlitz et al., Bio/Technology 8:651-654 (1990).

In addition to microorganisms and plants, mammalian cell culture canalso be used to express and produce the polypeptides of the presentinvention (see Winnacker, “From Genes to Clones”, VCH Publishers, NewYork (1987)). Mammalian cells are actually preferred, because a numberof suitable host cell lines capable of secreting intact immunoglobulinshave been developed in the art, and include the CHO cell lines, variousCOS cell lines, HeLa cells, preferably myeloma cell lines, etc., ortransformed B-cells or hybridomas. Expression vectors for these cellscan include expression control sequences, such as an origin ofreplication, a promoter, an enhancer (Queen et al., Immunol. Rev.89:49-68 (1986)), and necessary processing information sites, such asribosome binding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Preferred expression controlsequences are promoters derived from immunoglobulin genes, SV40,adenovirus, bovine papilloma virus, cytomegalovirus and the like.Generally, a selectable marker, such as a neo expression cassette, isincluded in the expression vector.

The present invention provides for a method of making an agent withaltered FcRn binding affinity and/or serum half-life by conjugating orotherwise binding of that agent to a moiety identified as having anincreased or reduced serum half-life through its interaction with FcRn.Such a moiety includes, but is not limited to, a modified IgG or amodified partial or full heavy chain comprising the amino acidsubstitutions described herein. Such agents would include, but are notlimited to, antibodies, fragments of antibodies, hormones, receptorligands, immunotoxins, therapeutic drugs of any kind, T-cell receptorbinding antigens, and any other agents that may be bound to theincreased serum half-life moieties of the present invention. To create afusion protein with an altered in vivo stability, DNA segments encodingsuch proteins may be operatively incorporated into a recombinant vector,in frame with the constant region of a modified antibody, whetherupstream or downstream, in a position so as to render the vector capableof expressing a fusion protein comprising such a protein operably linkedwith the constant region. Techniques for the manipulation of DNAsegments in this manner, for example, by genetic engineering usingrestriction endonucleases, will be known to those of skill in the art inlight of both the present disclosure and references such as Sambrook andRussell, supra.

The above method is proposed for use in the generation of a series oftherapeutic compounds with improved biological stability. Such compoundsinclude, for example, interleukin-2, insulin, interleukin-4, andinterferon gamma, or even T cell receptors. The recombinant Fc domainsof this invention are also contemplated to be of use in stabilizing awide range of drugs, which would likely alleviate the need for theirrepeated administration. However, the present methods are not limitedsolely to the production of proteins for human administration, and maybe employed to produce large quantities of any protein with increasedstability, such as may be used, for example, in immunization protocols,in animal treatment by veterinarians, or in rodent in vivo therapymodels.

III. Uses of Modified Antibodies with Altered FcRn Binding Affinityand/or Serum Half-lives

The present invention provides for a composition comprising the modifiedantibodies described herein and a pharmaceutically acceptable carrier.The compositions for parenteral administration commonly comprise asolution of the antibody or a cocktail thereof dissolved in anacceptable carrier, preferably an aqueous carrier. A variety of aqueouscarriers can be used, e.g., water, buffered water, 0.4% saline, 0.3%glycine and the like. These solutions are sterile and generally free ofparticulate matter. The compositions can contain pharmaceuticallyacceptable auxiliary substances as required to approximate physiologicalconditions such as pH adjusting and buffering agents, toxicity adjustingagents and the like, for example sodium acetate, sodium chloride,potassium chloride, calcium chloride, sodium lactate. The concentrationof the antibodies in these formulations can vary widely, i.e., from lessthan about 0.01%, usually at least about 0.1% to as much as 5% by weightand are selected primarily based on fluid volumes, and viscosities inaccordance with the particular mode of administration selected.

A typical composition for intravenous infusion can be made up to contain250 ml of sterile Ringer's solution, and 10 mg to 100 mg of antibody(see “Remington's Pharmaceutical Science”, 15^(th) ed., Mack PublishingCompany, Easton, Pa. (1980)).

The modified antibodies in the present invention can be used for variousnon-therapeutic purposes. They may be used as an affinity purificationagent. They may also be useful in diagnostic assays, such as detectingexpression of an antigen of interest in specific cells, tissues, orserum. For diagnostic applications, the antibodies typically will belabeled with a detectable moiety, including radioisotopes, fluorescentlabels, and various enzyme substrate labels. The antibodies may also beemployed in any known assay method, such as competitive binding assays,direct and indirect sandwich assays, and immunoprecipitation assays. Theantibodies may also be used for in vivo diagnostic assays. Generally,the antibodies are labeled with a radionucleotide so that the antigen orcell expressing it can be localized using immunoscintigraphy.

Kits can also be supplied for use with the modified antibodies in theprotection against or detection of a cellular activity or for thepresence of a selected cell surface receptor or the diagnosis ofdisease. Thus, the subject composition of the present invention may beprovided, usually in a lyophilized form in a container, either alone orin conjunction with additional antibodies specific for the desired celltype. The modified antibodies, which may be conjugated to a label ortoxin, or unconjugated, are included in the kits with buffers, such asTris, phosphate, carbonate, etc., stabilizers, biocides, inert proteins,e.g., serum albumin, or the like, and a set of instructions for use.Generally, these materials will be present in less than about 5% wt.based on the amount of active antibody, and usually present in totalamount of at least about 0.001% wt. based again on the antibodyconcentration. Frequently, it will be desirable to include an inertextender or excipient to dilute the active ingredients, where theexcipient may be present in from about 1 to 99% wt. of the totalcomposition. Where a second antibody capable of binding to the modifiedantibody is employed in an assay, this will usually be present in aseparate vial. The second antibody is typically conjugated to a labeland formulated in an analogous manner with the antibody formulationsdescribed above.

The modified antibodies have various therapeutic applications. Themodified antibodies may be used to treat a patient suffering from, orpredisposed to, a disease or disorder, who could benefit fromadministration of the modified antibodies. The conditions that can betreated with the antibodies include cancer; inflammatory conditions suchas asthma; autoimmune diseases; and viral infections, etc.

The cancers that can be treated by the antibodies described hereininclude, but are not limited to, breast cancer, squamous cell cancer,small cell lung cancer, non-small cell lung cancer, gastrointestinalcancer, pancreatic cancer, glioblastoma, cervical cancer, ovariancancer, bladder cancer, hepatoma, colon cancer, colorectal cancer,endometrial carcinoma, salivary gland carcinoma, kidney cancer, livercancer, prostate cancer, vulval cancer, thyroid cancer, hepaticcarcinoma, and various types of head and neck cancer.

The autoimmune diseases include, but are not limited to, Addison'sdisease, autoimmune diseases of the ear, autoimmune diseases of the eyesuch as uveitis, autoimmune hepatitis, Crohn's disease, diabetes (TypeI), epididymitis, glomerulonephritis, Graves' disease, Guillain-Barresyndrome, Hashimoto's disease, hemolytic anemia, systemic lupuserythematosus, multiple sclerosis, myasthenia gravis, pemphigusvulgaris, psoriasis, rheumatoid arthritis, sarcoidosis, scleroderma,Sjogren's syndrome, spondyloarthropathies, thyroiditis, ulcerativecolitis, and vasculitis.

The modified antibodies with reduced serum half-lives in the presentinvention may be used in the treatment of diseases or disorders wheredestruction or elimination of tissue or foreign microorganisms isdesired. For example, the antibody may be used to treat cancer;inflammatory disorders; infections; and other conditions where removalof tissue is desired. The antibody would be generally useful in that thequicker biological clearance times would result in reducedimmunogenicity of any antibody administered. Other applications wouldinclude antibody-based imaging regimens, antibody-based drug removal, orcreation of immunotoxins with a shorter life.

The modified antibodies with increased serum half-lives may be ananti-tissue factor (TF) antibody, anti-IgE antibody, and anti-integrinantibody. The desired mechanism of action may be to blockligand-receptor binding pairs. The modified antibodies with increasedserum half-lives may also be agonist antibodies. The antibodies can alsobe used as therapeutic agents such as vaccines. The dosage and frequencyof immunization of such vaccines will be reduced due to the extendedserum half-lives of the antibodies.

The compositions comprising the present antibodies are administered byany suitable means, including parenteral subcutaneous, intraperitoneal,intrapulmonary, and intranasal, and if desired for localimmunosuppressive treatment, intralesional administration. Parenteralinfusions include intramuscular, intravenous, intraarterial,intraperitoneal, or subcutaneous administration. In addition, theantibodies are suitably administered by pulse infusion, particularlywith declining doses of antibodies.

The compositions containing the present antibodies or a cocktail thereofcan be administered for prophylactic and/or therapeutic treatments. Intherapeutic application, compositions are administered to a patientalready affected by the particular disease, in an amount sufficient tocure or at least partially arrest the condition and its complications.An amount adequate to accomplish this is defined as a “therapeuticallyeffective dose.” Amounts effective for this use will depend upon theseverity of the condition and the general state of the patient's ownimmune system, but generally range from about 0.01 to about 100 mg ofmodified antibody per dose, with dosages of 1 to 10 mg per patient beingmore commonly used.

In prophylactic applications, compositions containing the modifiedantibodies or a cocktail thereof are administered to a patient notalready in a disease state to enhance the patient's resistance. Such anamount is defined to be a “prophylactically effective dose.” In thisuse, the precise amounts again depend upon the patient's state of healthand general level of immunity, but generally range from 0.1 to 100 mgper dose, especially dosages of 1 to 10 mg per patient.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of the mutant antibodies of this invention sufficient toeffectively treat the patient.

The following examples are offered by way of illustration and not by wayof limitation. The disclosure of all citations in the specification isexpressly incorporated herein by reference.

EXAMPLES Example 1

This example describes the antibody expression vectors used in thepresent invention.

The components of the heavy chain expression plasmid pVAg2M3-OST577, aderivative of the M3 variant of pVg2.D.Tt (Cole et al., J. Immunol.159:3613-3621 (1997)), are as follows. As shown in FIG. 5A, proceedingclockwise from the EcoRI site, the heavy chain unit begins with thehuman cytomegalovirus (hCMV) major immediate early (IE) promoter andenhancer (Boshart et al., Cell 41:521-530 (1985)) as an EcoRI-XbaIfragment. The hCMV region is followed by the OST577 V_(H) region as anXbaI fragment, including signal sequence, J segment, and splice donorsequence. The V_(H) region is followed by a modified genomic DNAfragment containing the human gamma-2M3 heavy chain constant region(Cole et al., op. cit.) as an XbaI-BamHI fragment, including the C_(H)1,hinge (H), C_(H)2, and C_(H)3 exons with the intervening introns, partof the intron preceding C_(H)1, and a polyadenylation (polyA) signal formRNA processing following C_(H)3, followed by the transcriptionalterminator of the human complement gene C2 (Ashfield et al., EMBO J.10:4197-4207 (1991)) as a BamHI-EcoRI fragment. The heavy chain unit isfollowed by a gene encoding a mutant form of dihydrofolate reductase(dhfr), together with regulatory elements (enhancer, promoter, splicesignals, and polyA signal) from Simian Virus 40 (SV40) needed fortranscription. This region, which was taken as a BamHI-EcoRI fragmentfrom the plasmid pVg1 (Co et al., op. cit.), was modified by convertingthe BamHI site to an EcoRI site. Moving counterclockwise within thisunit from the original EcoRI site, first there is part of the plasmidpBR322 (Sutcliffe, Cold Spring Harbor Symp. Quant. Biol. 43:77-90(1979)) comprising the bacterial origin of replication and ampicillinresistance gene for selection in E. coli, except the bacterialreplication origin was replaced with the corresponding segment frompUC18 (Yanisch-Perron et al., Gene 33:103-119 (1985)) to increase thecopy number of the vector in bacterial hosts. Next, there is a segmentof SV40 (Reddy et al., Science 200:494-502 (1978)) containing the SV40enhancer and early promoter to ensure strong transcription initiation.This segment is followed by the coding sequence of the E. coli dhfr gene(Simonsen and Levinson, Proc. Natl. Acad. Sci. USA 80:2495-2499 (1983)).The dhfr gene is followed by an SV40 segment containing the small tantigen intron, which is believed to increase mRNA levels, and then theplasmid contains another SV40 segment containing a polyA signal forending the mRNA transcript.

The components of the heavy chain expression plasmid pVAg1.N-OST577, aderivative of pVg1 (Co et al., op. cit.), are as follows. As shown inFIG. 5B, proceeding clockwise from the EcoRI site, the heavy chain unitbegins with the same EcoRI-XbaI fragment containing the hCMV IE promoterand enhancer that was used in the pVAg2M3-OST577 vector, followed by theOST577 V_(H) region as an XbaI fragment. The V_(H) region is followed bya genomic DNA fragment containing the human gamma-1 heavy chain constantregion (Ellison et al., Nucleic Acids Res. 10:4071-4079 (1982)) as anXbaI-BamHI fragment, including the C_(H)1, hinge (H), C_(H)2, and C_(H)3exons with the intervening introns, part of the intron preceding C_(H)1,and a polyA signal for mRNA processing following C_(H)3. To facilitatesubsequent manipulations of the coding regions, overlap-extension PCRmutagenesis (Higuchi, op. cit.) was used to create an NheI site in theintron between the hinge and C_(H)2 exons. The heavy chain unit isfollowed by the same BamHI-EcoRI restriction fragment encoding dhfr,together with regulatory elements and a portion of plasmid pBR322, thatwas used in the pVAg2M3-OST577 vector.

The components of the light chain expression plasmid pVAλ2-OST577, aderivative of pVk (Co et al., op. cit.), are as follows. As shown inFIG. 6, proceeding clockwise from the EcoRI site, the light chain unitbegins with the same EcoRI-XbaI fragment containing the hCMV IE promoterand enhancer that was used in the heavy chain vectors, followed by theOST577 V_(L) region as an XbaI fragment, including signal sequence, Jsegment, and splice donor sequence. The V_(L) region is followed by agenomic DNA fragment containing the human lambda-1 light chain constantregion (Hieter et al., Nature 294:536-540 (1981)) as an XbaI-Sau3AIfragment that was modified by PCR to encode the human lambda-2 lightchain constant region (Hieter et al., ibid.), including the humanlambda-1 light chain intron, the human lambda-2 light chain constantregion exon (C_(λ)2) and a portion of the 3′ untranslated region fromthe human lambda-2 light chain, and a polyA signal for mRNA processingfrom the human lambda-1 light chain. The light chain gene is followed bya gene encoding xanthine guanine phosphoribosyl transferase (gpt),together with regulatory elements from SV40 needed for transcription.The function of this region, which was taken as a BamHI-EcoRI fragmentfrom the plasmid pSV2-gpt (Mulligan and Berg, op. cit.), is to provide aselectable drug-resistance marker after transfection of the plasmid intomammalian cells. Moving counterclockwise within this unit from the EcoRIsite, first there is part of the plasmid pBR322 (Sutcliffe, op. cit.)comprising the bacterial origin of replication and ampicillin resistancegene for selection in E. coli, except the bacterial replication originwas replaced with the corresponding segment from pUC18 (Yanisch-Perronet al., op. cit.) to increase the copy number of the vector in bacterialhosts. Next, there is a segment of SV40 (Reddy et al., op. cit.)containing the SV40 enhancer and early promoter, to ensure strongtranscription initiation. This segment is followed by the codingsequence of the E. coli gpt gene (Richardson et al., Nucleic Acids Res.11:8809-8816 (1983)). The gpt gene is followed by an SV40 segmentcontaining the small t antigen intron, which is believed to increasemRNA levels, and then the plasmid contains another SV40 segmentcontaining a polyA signal for ending the mRNA transcript.

The components of the heavy chain expression plasmid pVAg2M3-Hu1D10 (seeFIG. 7A) are identical to those of pVAg2M3-OST577 from which it wasderived, except the OST577 V_(H) region was replaced with the Hu1D10V_(H) region from plasmid pHu1D10.IgG1.rgpt.dE (Kostelny et al. (2001),op. cit.). The components of the heavy chain expression plasmidpVAg1.N-Hu1D10 (see FIG. 7B) are identical to those of pVAg1.N-OST577from which it was derived, except the OST577 V_(H) region was replacedwith the Hu1D10 V_(H) region from plasmid pHu1D10.IgG1.rgpt.dE (Kostelnyet al., ibid).

The components of the heavy chain expression plasmid pHuHCg3.Tt.D-Hu1D10(see FIG. 7C) are identical to those of pVg2.D.Tt (Cole et al., op.cit.), except the XbaI-BamHI fragment containing the human gamma-2M3heavy chain constant region was replaced with the Hu1D10 V_(H) regionfrom plasmid pHu1D10.IgG1.rgpt.dE (Kostelny et al. (2001), op. cit.) asan XbaI fragment, followed by a genomic DNA fragment containing thehuman gamma-3 heavy chain constant region (Huck et al., Nucleic AcidsRes. 14:1779-1789 (1986)) as an Xba-BamHI fragment, including theC_(H)1, four hinge (H), C_(H)2, and C_(H)3 exons with the interveningintrons, part of the intron preceding C_(H)1, and a polyadenylation(polyA) signal for mRNA processing following C_(H)3. The components ofthe heavy chain expression plasmid pHuHCg4.Tt.D-Hu1D10 (see FIG. 7D) areidentical to those of pHuHCg3.Tt.D-Hu1D10, except the human gamma-3heavy chain constant region was replaced with a genomic DNA fragmentcontaining the human gamma-4 heavy chain constant region (Ellison etal., op. cit.) as an XbaI-BamHI fragment, including the C_(H)1, hinge(H), C_(H)2, and C_(H)3 exons with the intervening introns, part of theintron preceding C_(H)1, and a polyadenylation (polyA) signal for mRNAprocessing following C_(H)3.

The components of the light chain expression plasmid pVk-Hu1D10, aderivative of pVk (Co et al., op. cit.), are as follows. As shown inFIG. 8, proceeding clockwise from the EcoRI site, the light chain unitbegins with the same EcoRI-XbaI fragment containing the hCMV IE promoterand enhancer that was used in the heavy chain vectors, followed by theHu1D10 V_(L) region (Kostelny et al. (2001), op. cit.) as an XbaIfragment, including signal sequence, J segment, and splice donorsequence. The V_(L) region is followed by a genomic DNA fragmentcontaining the human kappa light chain constant region (Hieter et al.,Cell 22:197-207 (1980)) as an XbaI-BamHI fragment, including the humankappa light chain constant region exon (C_(κ)), part of the intronpreceding C_(κ), and a polyA signal for mRNA processing following C_(κ).The light chain unit is followed by the same BamHI-EcoRI fragmentencoding gpt, together with regulatory elements needed for transcriptionand a portion of plasmid pBR322, that was used in the pVk vector.

Example 2

This example describes the plasmids used in the present invention.

The OST577 heavy and light chain cDNAs were cloned in their entirety byPCR from a trioma cell line expressing human monoclonal anti-HBVantibody OST577 (Ehrlich et al., op. cit.). The heavy and light chainvariable regions were converted by PCR into miniexons, flanked by XbaIsites at both ends, comprising the signal sequences, V, (D), and Jsegments, splice donor sequences, and a portion of the correspondingintrons, as outlined in Co et al., supra. The expression vectorpVAg2M3-OST577 (see FIG. 5A), a derivative of the M3 variant ofpVg2.D.Tt (Cole et al., op. cit.), was constructed by replacing the XbaIfragment containing the OKT3-V_(H) miniexon with the OST577-V_(H)miniexon. Then the PciI-FspI fragment containing the bacterialreplication origin was replaced with the corresponding PciI-FspIfragment from pUC18 (Yanisch-Perron et al., op. cit.) to increase thecopy number of the vector in bacterial hosts. The expression vectorpVAg1.N-OST577 (see FIG. 5B), a derivative of pVg1 (Co et al., op.cit.), was constructed by inserting an XbaI fragment containing theOST577-V_(H) miniexon into the unique XbaI site of pVg1, modifying thehinge-C_(H)2 intron by overlap-extension PCR (Higuchi, op. cit.) tocreate a unique NheI site, and replacing the HindIII-XhoI fragmentcontaining the bacterial replication origin with the correspondingHindIII-XhoI fragment from pVAg2M3-OST577 to increase the copy number ofthe vector in bacterial hosts.

The expression vector pVλ2-OST577, a derivative of pVk (Co et al., op.cit.), was constructed by first replacing the XbaI-BamHI fragment of pVkcontaining the genomic human kappa constant region with an XbaI-BglIIPCR product containing the genomic human lambda-1 constant region. Thecoding region and a portion of the 3′ untranslated region were replacedwith the corresponding fragment from the OST577 light chain cDNA by PCR,essentially yielding a genomic human lambda-2 constant region exon.Finally, the OST577-V_(L) miniexon was inserted into the XbaI site ofthe vector. The expression vector pVAλ2-OST577 (see FIG. 6), aderivative of pVλ2-OST577, was constructed by replacing the SapI-FspIfragment containing the bacterial replication origin with thecorresponding SapI-FspI fragment from pVAg2M3-OST577 to increase thecopy number of the vector in bacterial hosts.

The expression vector pVAg2M3-Hu1D10 (see FIG. 7A) was constructed byreplacing the XhoI-XbaI fragment from plasmid pVAg2M3-OST577, containingthe hCMV promoter and enhancer (Boshart et al., op. cit.) and the OST577V_(H) region, with the corresponding XhoI-XbaI fragment from plasmidpHu1D10.IgG1.rgpt.dE (Kostelny et al. (2001), op. cit.) containing thehCMV promoter and enhancer and the Hu1D10 V_(H) region. The expressionvector pVAg1.N-Hu1D10 (see FIG. 7B) was constructed by replacing theXhoI-XbaI fragment from plasmid pVAg1.N-OST577, containing the hCMVpromoter and enhancer (Boshart et al., op. cit.) and the OST577 V_(H)region, with the corresponding XhoI-XbaI fragment from plasmidpHu1D10.IgG1.rgpt.dE (Kostelny et al. (2001), op. cit.) containing thehCMV promoter and enhancer and the Hu1D10 V_(H) region.

The expression vector pHuHC.g3.Tt.D-Hu1D10 (see FIG. 7C) was constructedby replacing the XhoI-BamHI fragment from the M3 variant of pVg2.D.Tt(Cole et al., op. cit.), containing the hCMV promoter and enhancer andthe human gamma-2M3 heavy chain constant region, with a XhoI-XbaIfragment from plasmid pHu1D10.IgG1.rgpt.dE (Kostelny et al. (2001), op.cit.) containing the hCMV promoter and enhancer and the Hu1D10 V_(H)region, and an XbaI-BamHI fragment containing the human gamma-3 heavychain constant region (Huck et al., op. cit.), respectively. Theexpression vector pHuHC.g4.Tt.D-Hu1D10 (see FIG. 7D) was constructed byreplacing the XhoI-BamHI fragment from the M3 variant of pVg2.D.Tt (Coleet al., op. cit.), containing the hCMV promoter and enhancer and the thehuman gamma-2M3 heavy chain constant region, with a XhoI-XbaI fragmentfrom plasmid pHu1D10.IgG1.rgpt.dE (Kostelny et al. (2001), op. cit.)containing the hCMV promoter and enhancer and the Hu1D10 V_(H) region,and an XbaI-BamHI fragment containing the human gamma-4 heavy chainconstant region (Ellison et al., op. cit.), respectively.

The expression vector pVk-Hu1D10 (see FIG. 8) was constructed byreplacing the XhoI-XbaI fragment from plasmid pVk (Co et al., op. cit.),containing the hCMV promoter and enhancer (Boshart et al., op. cit.),with the XhoI-XbaI fragment from plasmid pHu1D10.IgG1.rgpt.dE (Kostelnyet al. (2001), op. cit.) containing the hCMV promoter and enhancer andthe Hu1D10 V_(L) region.

The base expression vector pDL172, a derivative of pVk.rg (Cole et al.,op. cit.), was constructed by replacing the XbaI-SphI fragmentcontaining the genomic human kappa constant region with an XbaI-SphIfragment comprised of an XbaI-NheI fragment containing the N-terminalportion of the M195 heavy chain signal sequence (Co et al., op. cit.), a0.7 kb NheI-AgeI fragment, a synthetic AgeI-EagI fragment encoding ahuman c-myc decapeptide, flanked by linker peptides, that is recognizedby mouse monoclonal antibody 9E10 (Evan et al., Mol. Cell. Biol.5:3610-3616 (1985)), followed by the GPI linkage signal from human decayaccelerating factor (Caras et al., Nature 325:545-549 (1987)), and anEagI-SphI fragment containing the polyA signal of the humanimmunoglobulin gamma-1 gene (Ellison et al., op. cit.).

Human beta-2 microglobulin (β2m) and the extracellular domains of thehuman neonatal Fc receptor (FcRn) alpha chain were cloned by PCR from acDNA library prepared from human peripheral blood mononuclear cells. Thehuman FcRn alpha chain gene was modified by PCR to add a flanking NheIsite and the C-terminal portion of the M195 heavy chain signal sequence(Co et al., op. cit.) at the 5′ end, and a flanking AgeI site at the 3′end, and used to replace the NheI-AgeI fragment of pDL172, resulting inexpression vector pDL172+HuFcRn. The human β2m gene was modified by PCRto add flanking XbaI and SalI sites at the 5′ and 3′ ends, respectively,and to remove an internal EcoRI site. The resulting XbaI-SalI fragmentwas subcloned into an intermediate vector, flanked on its 5′ end by anEcoRI-XbaI fragment containing the hCMV IE promoter and enhancer(Boshart et al., op. cit.), and on its 3′ end by a SalI-BamHI fragmentcontaining the polyadenylation signal of the murine immunoglobulingamma-2a gene (Kostelny et al. (1992), op. cit.), followed by aBamHI-EcoRI fragment containing the transcriptional terminator of thehuman complement gene C2 (Ashfield et al., op. cit.). The resultingEcoRI-EcoRI fragment containing a functional human β2m transcriptionalunit was cloned into the unique EcoRI site of pDL172+HuFcRn, resultingin expression vector pDL172+HuFcRn+Hufβ2m, hereinafter referred to aspDL208 (see FIG. 9A).

Rhesus β2m and the extracellular domains of the rhesus FcRn alpha chainwere cloned by PCR from a cDNA library prepared from rhesus peripheralblood mononuclear cells. The rhesus β2m gene was modified by PCR to addflanking XbaI and SalI sites at the 5′ and 3′ ends, respectively, and toremove an internal EcoRI site. The resulting XbaI-SalI fragment wassubcloned into an intermediate vector, flanked on its 5′ end by anEcoRI-XbaI fragment containing the hCMV IE promoter and enhancer(Boshart et al., op. cit.), and on its 3′ end by a SalI-BamHI fragmentcontaining the polyadenylation signal of the murine immunoglobulingamma-2a gene (Kostelny et al. (1992), op. cit.), followed by aBamHI-EcoRI fragment containing the transcriptional terminator of thehuman complement gene C2 (Ashfield et al., op. cit.). The resultingEcoRI-EcoRI fragment containing a functional rhesus β2m transcriptionalunit was used to replace the EcoRI-EcoRI fragment (containing the humanβ2m transcriptional unit) of pDL172+HuFcRn+Huβ2m, resulting inpDL172+HuFcRn+Rhβ2m. The rhesus FcRn alpha chain gene was modified byPCR to add a flanking NheI site and the C-terminal portion of the M195heavy chain signal sequence (Co et al., op. cit.) at the 5′ end, and aflanking AgeI site at the 3′ end, and used to replace the NheI-AgeIfragment (containing the human FcRn alpha chain gene) ofpDL172+HuFcRn+Rhβ2m, resulting in expression vector pDL172+RhFcRn+Rhβ2m,hereinafter referred to as pDL410 (see FIG. 9B).

Example 3

This example describes mutagenesis of the Fc region of the human γ2M3heavy chain gene.

Molecular Modeling:

An initial model of the human Fc/FcRn complex was generated based on alow-resolution crystal structure of the rat Fc/FcRn complex (Burmeisteret al., Nature 372:379-383 (1994); RCSB protein databank code 1FRT).First, the rat β2m of the complex was replaced by superimposing thehuman β2m taken from a high-resolution crystal structure of the humanhistocompatibility antigen HLA-A2 (Saper et al. J. Mol. Biol.219:277-319 (1991); RCSB code 3HLA) in the same orientation as that ofthe rat β2m in the complex. Then, the alpha chain of the rat FcRn wasreplaced by superimposing the human alpha chain taken from ahigh-resolution crystal structure of human FcRn (West and Bjorkman,Biochemistry 29:9698-9708 (2000); RCSB code 1EXU) in the sameorientation as the rat alpha chain in the complex. Next, the ratresidues in the Fc of the complex were replaced with the correspondingresidues from the human IgG1 Fc (Kabat et al., op. cit.), and energyminimization calculations were done using the SEGMOD and ENCAD programs(Levitt, J. Mol. Biol. 226:507-533 (1992); Levitt, J. Mol. Biol.168:595-620 (1983)) to produce a model of the human IgG1 Fc/FcRncomplex. Finally, the human IgG1 Fc residues of the model were replacedwith the corresponding residues from the human IgG2M3 Fc (Cole et al.,op. cit.), and the energy minimization calculations were repeated toproduce a model of the human IgG2M3 Fc/FcRn complex, hereinafterreferred to as model 1.

A second model of the human IgG2M3 Fc/FcRn complex was generated asdescribed above, based on a model of the rat Fc/FcRn complex (Weng etal., J. Mol. Biol. 282:217-225 (1998); RCSB code 2FRT), hereinafterreferred to as model 2.

A third model was generated as described above, based on thehigh-resolution crystal structure of a heterodimeric rat Fc/FcRn complex(Martin et al., Mol. Cell 7:867-877 (2001); RCSB code 1|1A), hereinafterreferred to as model 3.

Mutagenesis:

The overlap-extension polymerase chain reaction (PCR) method (Higuchi,op. cit.) was used to generate random amino acid substitutions atpositions 250, 314, and 428 of the OST577-IgG2M3 heavy chain (numberedaccording to the EU index of Kabat et al., op. cit.). To generate randommutants at position 250, the mutagenesis primers JY24 (5′-GAC CTC AGGGGT CCG GGA GAT CAT GAG MNN GTC CTT GG-3′) (SEQ ID NO: 77) and JY25(5′-CTC ATG ATC TCC CGG ACC CCT GAG GTC-3′) (SEQ ID NO: 78) were used,where M=A or C, and N=A, C, G or T. The first round of overlap-extensionPCR used outside primer msc g2-1 (5′-CCA GCT CTG TCC CAC ACC G-3′) (SEQID NO: 79) and JY24 for the left-hand fragment, and outside primer kco8(5′-GCC AGG ATC CGA CCC ACT-3′) (SEQ ID NO: 80) and JY25 for theright-hand fragment. The PCR reaction was done using the Expand™ HighFidelity PCR System (Roche Diagnostics Corporation, Indianapolis, Ind.),following the manufacturer's recommendations, by incubating at 94° C.for 5 minutes, followed by 25 cycles of 94° C. for 5 seconds, 55° C. for5 seconds and 72° C. for 60 seconds, followed by incubating at 72° C.for 7 minutes in a GeneAmp® PCR System 9600 (Applied Biosystems®, FosterCity, Calif.). The PCR products were run on a low-melting point agarosegel, excised from the gel, and melted at 70° C. The second round of PCRto combine the left-hand and right-hand fragments was done as describedabove, using outside primers msc g2-1 and kco8, for 35 cycles. The finalPCR products were run on a low-melting point agarose gel and DNAfragments of the expected size were excised and purified using theQIAEX™II Gel Extraction Kit (QIAGEN®, Valencia, Calif.). The purifiedfragments were digested with PinAI and BamHI, gel-purified as describedabove, and cloned between the corresponding sites in pVAg2M3-OST577.

To generate the T2501 and T250L mutants, the mutagenesis primers KH4(5′-GAC CTC AGG GGT CCG GGA GAT CAT GAG AAK GTC CTT GG-3′) (SEQ ID NO:81) and KH3 (5′-CTC ATG ATC TCC CGG ACC CCT GAG GTC-3′) (SEQ ID NO: 82)were used, where K=G or T. To generate the T250C and T250G mutants, themutagenesis primers KH5 (5′-GAC CTC AGG GGT CCG GGA GAT CAT GAG GCM GTCCTT GG-3′) (SEQ ID NO: 83) and KH3 were used, where M=A or C. Togenerate the T250N and T250Q mutants, the mutagenesis primers KH6(5′-GAC CTC AGG GGT CCG GGA GAT CAT GAG NTK GTC CTT GG-3′) (SEQ ID NO:84) and KH3 were used, where K=G or T, and N=A, C, G or T. The firstround of PCR used outside primer msc g2-1 and KH4, KH5 or KH6 for theleft-hand fragment, and outside primer MGD-1 (5′-GCC AGG ATC CGA CCCACT-3′) (SEQ ID NO: 85) and KH3 for the right-hand fragment. The PCRreactions were done using the Expand™ High Fidelity PCR System (RocheDiagnostics Corporation) by incubating at 94° C. for 5 minutes, followedby 25 cycles of 94° C. for 5 seconds, 60° C. for 5 seconds and 72° C.for 60 seconds, followed by incubating at 72° C. for 7 minutes. The PCRproducts were run on a low-melting point agarose gel, excised from thegel, and melted at 70° C. The second round of PCR to combine theleft-hand and right-hand fragments was done as described above, usingoutside primers msc g2-1 and MGD-1, by incubating at 94° C. for 5minutes, followed by 35 cycles of 94° C. for 5 seconds, 60° C. for 5seconds and 72° C. for 105 seconds, followed by incubating at 72° C. for7 minutes. The final PCR products were run on a low-melting pointagarose gel and DNA fragments of the expected size were excised andpurified using the QIAquick™ Gel Extraction Kit (QIAGEN®). The purifiedfragments were digested with PinAI and BamHI, gel-purified as describedabove, and cloned between the corresponding sites in pVAg2M3-OST577.

To generate random mutants at position 314, the mutagenesis primerskco78 (5′-ACC GTT GTG CAC CAG GAC TGG NNK AAC GGC AAG GAG-3′) (SEQ IDNO: 86) and kco79 (5′-CCA GTC CTG GTG CAC AAC GG-3′) (SEQ ID NO: 87)were used, where K=G or T, and N=A, C, G or T. The first round of PCRused outside primer ks g2-5 (5′-CTC CCG GAC CCC TGA GGT C-3′) (SEQ IDNO: 88) and kco79 for the left-hand fragment, and outside primer kco8and kco78 for the right-hand fragment. All subsequent steps were done asdescribed above for position 250 random mutagenesis, except the secondround of PCR used outside primers ks g2-5 and kco8, and the final PCRfragments were digested with PmlI and BamHI and cloned into thecorresponding sites in pVAg2M3-OST577.

To generate the L314I mutant, the mutagenesis primers MGD-10 (5′-ACC GTTGTG CAC CAG GAC TGG ATC AAC GGC AAG GA-3′) (SEQ ID NO: 89) and kco79were used. To generate the L314Y mutant, the mutagenesis primers MGD-11(5′-ACC GTT GTG CAC CAG GAC TGG TAT AAC GGC AAG GA-3′) (SEQ ID NO: 90)and kco79 were used. To generate the L314H mutant, the mutagenesisprimers MGD-12 (5′-ACC GTT GTG CAC CAG GAC TGG CAC AAC GGC AAG GA-3′)(SEQ ID NO: 91) and kco79 were used. To generate the L314M mutant, themutagenesis primers MGD-13 (5′-ACC GTT GTG CAC CAG GAC TGG ATG AAC GGCAAG GA-3′) (SEQ ID NO: 92) and kco79 were used. To generate the L314Nmutant, the mutagenesis primers MGD-14 (5′-ACC GTT GTG CAC CAG GAC TGGAAT AAC GGC AAG GA-3′) (SEQ ID NO: 93) and kco79 were used. The firstround of PCR used outside primer jt240 (5′-GGA CAC CTT CTC TCC TCC C-3′)(SEQ ID NO: 94) and kco79 for the left-hand fragment, and outside primerkco41 (5′-ATT CTA GTT GTG GTT TGT CC-3′) (SEQ ID NO: 95) and MGD-10,MGD-11, MGD-12, MGD-13 or MGD-14 for the right-hand fragment. The PCRreactions were done using the Expand™ High Fidelity PCR System (RocheDiagnostics Corporation) by incubating at 94° C. for 5 minutes, followedby 25 cycles of 94° C. for 5 seconds, 60° C. for 5 seconds and 72° C.for 60 seconds, followed by incubating at 72° C. for 7 minutes. The PCRproducts were run on a low-melting point agarose gel, excised from thegel, and melted at 70° C. The second round of PCR to combine theleft-hand and right-hand fragments was done as described above, usingoutside primers jt240 and kco41, by incubating at 94° C. for 5 minutes,followed by 35 cycles of 94° C. for 5 seconds, 60° C. for 5 seconds and72° C. for 90 seconds, followed by incubating at 72° C. for 7 minutes.The final PCR products were run on a low-melting point agarose gel andDNA fragments of the expected size were excised and purified using theQIAquick™ Gel Extraction Kit (QIAGEN®). The purified fragments weresubcloned in pCR®4Blunt-TOPO® (Invitrogen™, Carlsbad, Calif.), thendigested with PmlI and BamHI, gel-purified as described above, andcloned between the corresponding sites in pVAg2M3-OST577.

To generate random mutants at position 428, the mutagenesis primers JY22(5′-GAA CGT CTT CTC ATG CTC CGT GNN KCA TGA GGC TCT G-3′) (SEQ ID NO:96) and JY23 (5′-CAC GGA GCA TGA GAA GAC GTT C-3′) (SEQ ID NO: 97) wereinitially used, where K=G or T, and N=A, C, G or T. The first round ofPCR used outside primer ks g2-5 and JY23 for the left-hand fragment, andoutside primer kco8 and JY22 for the right-hand fragment. All subsequentsteps were done as described above for position 314 random mutagenesis.

To generate additional random mutants at position 428, the mutagenesisprimers MGD-2 (5′-GTG TAG TGG TTG TGC AGA GCC TCA TGM NNC ACG GAG CATGAG AAG-3′) (SEQ ID NO: 98) and KH1 (5′-CAT GAG GCT CTG CAC AAC CAC TACAC-3′) (SEQ ID NO: 99) were subsequently used, where M=A or C, and N=A,C, G or T. The first round of PCR used outside primer msc g2-1 and MGD-2for the left-hand fragment, and outside primer MGD-1 and KH1 for theright-hand fragment. The PCR reaction was done using the Expand™ HighFidelity PCR System (Roche Diagnostics Corporation) by incubating at 94°C. for 5 minutes, followed by 25 cycles of 94° C. for 5 seconds, 60° C.for 5 seconds and 72° C. for 90 seconds, followed by incubating at 72°C. for 7 minutes. The PCR products were run on a low-melting pointagarose gel, excised from the gel, and melted at 70° C. The second roundof PCR to combine the left-hand and right-hand fragments was done asdescribed above, using outside primers msc g2-1 and MGD-1, by incubatingat 94° C. for 5 minutes, followed by 35 cycles of 94° C. for 5 seconds,60° C. for 5 seconds and 72° C. for 75 seconds, followed by incubatingat 72° C. for 7 minutes. The final PCR products were run on alow-melting point agarose gel and DNA fragments of the expected sizewere excised and purified using the QIAquick™ Gel Extraction Kit(QIAGEN®). The purified fragments were digested with PinAI and BamHI,gel-purified as described above, and cloned between the correspondingsites in pVAg2M3-OST577.

To generate the M428E mutant, the mutagenesis primers MGD-8 (5′-GTG TAGTGG TTG TGC AGA GCC TCA TGT TCC ACG GAG CAT GAG AAG-3′) (SEQ ID NO: 100)and KH1 were used. The first round of PCR used outside primer msc g2-1and MGD-8 for the left-hand fragment, and outside primer MGD-1 and KH1for the right-hand fragment. All subsequent steps were done as describedabove for position 428 random mutagenesis.

The T250E/M428F double mutant was generated by replacing the PmlI-BamHIfragment in the pVAg2M3-OST577 plasmid containing the T250E mutationwith the corresponding PmlI-BamHI fragment from the pVAg2M3-OST577plasmid containing the M428F mutation. The T250Q/M428F and T250Q/M428Ldouble mutants were generated by replacing the PmlI-BamHI fragment inthe pVAg2M3-OST577 plasmid containing the T250Q mutation with thecorresponding PmlI-BamHI fragments from the pVAg2M3-OST577 plasmidscontaining the M428F and M428L mutations, respectively.

Several amino acid substitutions were also created at positions 250 and428 of the Hu1D10-IgG2M3 heavy chain. To generate the M428L mutant, theXhoI-XbaI fragment from plasmid pVAg2M3-OST577 (M428L), containing thehCMV promoter and enhancer (Boshart et al., op. cit.) and the OST577V_(H) region, was replaced with the corresponding XhoI-XbaI fragmentfrom plasmid pHu1D10.IgG1.rgpt.dE (Kostelny et al. (2001), op. cit.)containing the hCMV promoter and enhancer and the Hu1D10 V_(H) region.To generate the T250Q/M428L mutant, the XhoI-XbaI fragment from plasmidpVAg2M3-OST577 (T250Q/M428L), containing the hCMV promoter and enhancer(Boshart et al., op. cit.) and the OST577 V_(H) region, was replacedwith the corresponding XhoI-XbaI fragment from plasmidpHu1D10.IgG1.rgpt.dE (Kostelny et al. (2001), op. cit.) containing thehCMV promoter and enhancer and the Hu1D10 V_(H) region.

Plasmid DNA was prepared using the QIAprep™ Spin Miniprep Kit (QIAGEN®),and nucleotide substitutions were identified by sequencing. Large-scaleplasmid DNA preparations were made using the EndoFree® Plasmid Maxi Kit(QIAGEN®). The coding regions of the OST577-IgG2M3 expression plasmidswere verified by nucleotide sequencing.

Results:

In order to isolate human IgG mutants with higher or lower affinity tothe neonatal Fc receptor (FcRn), which would be expected to have alteredserum half-lives, random amino acid substitutions were generated atpositions 250, 314, and 428 of the human γ2M3 heavy chain (numberedaccording to the EU index of Kabat et al., op. cit.). These threepositions were chosen based on computer modeling of a complex of thehuman IgG2M3 Fc and human FcRn (see models 1, 2, and 3, which aredescribed earlier in the Example), which was deduced from the X-raycrystal structure of the rat Fc/FcRn complex (Burmeister et al., op.cit.). Although the wild-type amino acids at positions 250, 314, and 428are located near the Fc/FcRn interface, these residues do not appear todirectly contribute to the pH-dependent interaction between Fc and FcRn.Therefore, amino acid substitutions at these positions may increase (ordecrease) the affinity of Fc for FcRn while maintaining pH-dependentbinding. Among the single mutants generated by PCR-based mutagenesiswere 19 mutants that converted the wild-type T at position 250 to A, C,D, E, F, G, H, I, K, L, M, N, P, Q, R, S, V, W, or Y; 19 mutants thatconverted the wild-type L at position 314 to A, C, D, E, F, G, H, I, K,M, N, P, Q, R, S, T, V, W, or Y; and 19 mutants that converted thewild-type M at position 428 to A, C, D, E, F, G, H, I, K, L, N, P, Q, R,S, T, V, W, or Y (see Table 1). Some of the single mutants withincreased binding to FcRn were combined to generate several doublemutants, including T250E/M428F, T250Q/M428F, and T250Q/M428L.

Example 4

This example describes mutagenesis of the Fc region of the human γ1heavy chain gene.

Mutagenesis:

The overlap-extension PCR method (Higuchi, op. cit.) was used togenerate amino acid substitutions at positions 250 and 428 of theOST577-IgG1 heavy chain (numbered according to the EU index of Kabat etal., op. cit.). To generate the T250E mutant, the mutagenesis primersJX076 (5′-AAC CCA AGG ACG AAC TCA TGA TCT CCC G-3′) (SEQ ID NO: 101) andJX077 (5′-GGA GAT CAT GAG TTC GTC CTT GGG TTT TG-3′) (SEQ ID NO: 102)were used. The first round of overlap-extension PCR used outside primerJX080 (5′-CCT CAG CTC GGA CAC CTT CTC-3′) (SEQ ID NO: 103) and JX077 forthe left-hand fragment, and outside primer NT244 (5′-GCC TCC CTC ATG CCACTC A-3′) (SEQ ID NO: 104) and JX076 for the right-hand fragment. ThePCR reaction was done using the Expand™ High Fidelity PCR System (RocheDiagnostics Corporation), following the manufacturer's recommendations,by incubating at 94° C. for 5 minutes, followed by 35 cycles of 94° C.for 20 seconds, 55° C. for 20 seconds and 72° C. for 90 seconds,followed by incubating at 72° C. for 7 minutes in a GeneAmp® PCR System9600 (Applied Biosystems®). The PCR products were run on a low-meltingpoint agarose gel, excised from the gel, and melted at 70° C. The secondround of PCR to combine the left-hand and right-hand fragments was doneas described above, using outside primers JX080 and NT244, for 35cycles. The final PCR products were run on a low-melting point agarosegel and DNA fragments of the expected size were excised and purifiedusing the QIAquick™II Gel Extraction Kit (QIAGEN®). The purifiedfragments were digested with NheI and EagI, gel-purified as describedabove, and cloned between the corresponding sites in pVAg1.N-OST577.

To generate the T250D mutant, the mutagenesis primers JX087 (5′-AAC CCAAGG ACG ACC TCA TGA TCT CCC G-3′) (SEQ ID NO: 105) and JX088 (5′-GGA GATCAT GAG GTC GTC CTT GGG TTT TG-3′) (SEQ ID NO: 106) were used. The firstround of PCR used outside primer JX080 and JX088 for the left-handfragment, and outside primer NT244 and JX087 for the right-handfragment. All subsequent steps were done as described above.

To generate the M428F mutant, the mutagenesis primers JX078 (5′-CTC ATGCTC CGT GTT CCA TGA GGC TCT GC-3′) (SEQ ID NO: 107) and JX079 (5′-AGAGCC TCA TGG AAC ACG GAG CAT GAG-3′) (SEQ ID NO: 108) were used. Thefirst round of PCR used outside primer JX080 and JX079 for the left-handfragment, and outside primer NT244 and JX078 for the right-handfragment. All subsequent steps were done as described above.

To generate the M428L mutant, the mutagenesis primers JXM428L1 (5′-CTCATG CTC CGT GTT GCA TGA GGC TCT GC-3′) (SEQ ID NO: 109) and JXM428L2(5′-AGA GCC TCA TGC AAC ACG GAG CAT GAG-3′) (SEQ ID NO: 110) were used.The first round of PCR used outside primer JX080 and JXM428L2 for theleft-hand fragment, and outside primer NT244 and JXM428L1 for theright-hand fragment. All subsequent steps were done as described above.

To generate the T250Q mutant, the mutagenesis primers JXT250Q1 (5′-AACCCA AGG ACC AAC TCA TGA TCT CCC G-3′) (SEQ ID NO: 111) and JXT250Q2(5′-GGA GAT CAT GAG TTG GTC CTT GGG TTT TG-3′) (SEQ ID NO: 112) wereused. The first round of PCR used outside primer JX080 and JXT250Q2 forthe left-hand fragment, and outside primer NT244 and JXT250Q1 for theright-hand fragment. All subsequent steps were done as described above.

To generate the T250E/M428F double mutant, the mutagenesis primers JX076and JX077 were used to create the T250E mutation in a templatecontaining the M428F mutation. The first round of PCR used outsideprimer JX080 and JX077 for the left-hand fragment, and outside primerNT244 and JX076 for the right-hand fragment. All subsequent steps weredone as described above.

To generate the T250Q/M428L double mutant, the mutagenesis primersJXT250Q1 and JXT250Q2 were used to create the T250Q mutation, and themutagenesis primers JXM428L1 and JXM428L2 were used to create the M428Lmutation. The first round of PCR used outside primer JX080 and JXT250Q2to create the left-hand fragment, JXT250Q1 and JXM428L2 to create themiddle fragment, and outside primer NT244 and JXM428L1 to create theright-hand fragment. All subsequent steps were done as described above.

Several amino acid substitutions were also created at positions 250 and428 of the Hu1D10-IgG1 heavy chain. To generate the M428L mutant, theXhoI-XbaI fragment from plasmid pVAg1.N-OST577 (M428L), containing thehCMV promoter and enhancer (Boshart et al., op. cit.) and the OST577V_(H) region, was replaced with the corresponding XhoI-XbaI fragmentfrom plasmid pHu1D10.IgG1.rgpt.dE (Kostelny et al. (2001), op. cit.)containing the hCMV promoter and enhancer and the Hu1D10 V_(H) region.To generate the T250Q/M428L mutant, the XhoI-XbaI fragment from plasmidpVAg1.N-OST577 (T250Q/M428L), containing the hCMV promoter and enhancer(Boshart et al., op. cit.) and the OST577 V_(H) region, was replacedwith the corresponding XhoI-XbaI fragment from plasmidpHu1D10.IgG1.rgpt.dE (Kostelny et al. (2001), op. cit.) containing thehCMV promoter and enhancer and the Hu1D10 V_(H) region.

Plasmid DNA was prepared using the QIAprep™ Spin Miniprep Kit (QIAGEN®),and nucleotide substitutions were confirmed by sequencing. Large-scaleplasmid DNA preparations were made using the EndoFree™ Plasmid Maxi Kit(QIAGEN®). The coding regions of the OST577-IgG1 expression plasmidswere verified by nucleotide sequencing.

Results:

In order to identify human IgG mutants with altered affinity to theneonatal Fc receptor (FcRn), which would be expected to have alteredserum half-lives, several amino acid substitutions were generated atpositions 250 and 428 of the human γ1 heavy chain (numbered according tothe EU index of Kabat et al., op. cit.). These two positions were chosenbased on the identification of mutations at these positions in the humanγ2M3 heavy chain that resulted in increased or decreased binding toFcRn. Although the wild-type amino acids at positions 250 and 428 arelocated near the Fc/FcRn interface, these residues do not appear todirectly contribute to the pH-dependent interaction between Fc and FcRn.Therefore, amino acid substitutions at these positions may increase (ordecrease) the affinity of Fc for FcRn while maintaining pH-dependentbinding. Both single and double mutants that exhibited increased bindingin the context of the human γ2M3 heavy chain were evaluated in the humanγ1 heavy chain, including the single mutants T250E, T250Q, M428F, andM428L, and the double mutants T250E/M428F and T250Q/M428L. A singlemutant that exhibited decreased binding in the context of the human γ2M3heavy chain (T250D) was also evaluated in the human γ1 heavy chain.

Example 5

This example describes the characterization of mutant IgG2M3 and IgG1antibodies.

Cell Culture:

Human kidney cell line 293-H (Life Technologies®, Rockville, Md.) wasmaintained in DMEM (BioWhittaker™, Walkersville, Md.) containing 10%Fetal Bovine Serum (FBS) (HyClone®, Logan, Utah), 0.1 mM MEMnon-essential amino acids (Invitrogen™) and 2 mM L-glutamine(Invitrogen™), hereinafter referred to as 293 medium, at 37° C. in a7.5% CO₂ incubator. For expression and purification of monoclonalantibodies after transient transfection, 293-H cells were incubated inDMEM containing 10% low-IgG FBS (HyClone®), 0.1 mM MEM non-essentialamino acids and 2 mM L-glutamine, hereinafter referred to as low-IgG 293medium. Mouse myeloma cell line Sp2/0 (American Type Culture Collection,Manassus, Va.) was maintained in DMEM containing 10% FBS and 2 mML-glutamine. For purification of monoclonal antibodies after stabletransfections, Sp2/0 cells were adapted to growth in Hybridoma-SFM(HSFM) (Life Technologies®).

Transient Transfections:

293-H cells were transiently cotransfected with the appropriate lightchain plasmid and the appropriate wild-type or one of the variousmutated heavy chain plasmids containing a single or double amino acidsubstitution at position 250, 314 or 428. For small-scale transienttransfections, approximately 1×10⁶ cells per transfection were plated ina 6-well plate in 3 ml of 293 medium and grown overnight to confluence.The next day, 2 μg of light chain plasmid and 2 μg of wild-type ormutated heavy chain plasmid were combined with 0.25 ml of HSFM. In aseparate tube, 10 μl of Lipofectamine™ 2000 reagent (Invitrogen™) and0.25 ml of HSFM were combined and incubated for 5 minutes at roomtemperature. The 0.25 ml Lipofectamine™ 2000-HSFM mixture was mixedgently with the 0.25 ml DNA-HSFM mixture and incubated at roomtemperature for 20 minutes. The medium covering the 293-H cells wasaspirated and replaced with low-IgG 293 medium, then thelipofectamine-DNA complexes were added dropwise to the cells, mixedgently by swirling, and the cells were incubated for 5-7 days at 37° C.in a 7.5% CO₂ incubator before harvesting the supernatants.

For large-scale transient transfections, approximately 7×10⁶ cells pertransfection were plated in a T-75 flask in 25 ml of 293 medium andgrown overnight to confluence. The next day, 12 μg of light chainplasmid and 12 μg of wild-type or mutated heavy chain plasmid werecombined with 1.5 ml of HSFM. In a separate tube, 60 μl ofLipofectamine™ 2000 reagent and 1.5 ml of HSFM were combined andincubated for 5 minutes at room temperature. The 1.5 ml Lipofectamine™2000-HSFM mixture was mixed gently with the 1.5 ml DNA-HSFM mixture andincubated at room temperature for 20 minutes. The medium covering the293-H cells was aspirated and replaced with low-IgG 293 medium, then thelipofectamine-DNA complexes were added dropwise to the cells, mixedgently by swirling, and the cells were incubated for 5-7 days at 37° C.in a 7.5% CO₂ incubator before harvesting the supernatants.

Antibody Concentration:

Supernatants from small-scale transient transfections were harvested bycentrifugation at ˜1,200 rpm for 5 minutes and sterile filtered using0.22 μm Millex®-GV microfilters (Millipore® Corporation, Bedford,Mass.). The samples were concentrated approximately 6-fold to 0.5 mlusing 6 ml Vivaspin® concentrators (50,000 MWCO) (Vivascience® AG,Hannover, Germany) by centrifugation at 3,000 rpm. Concentrated proteinsamples were resuspended in 5 ml of PBS, pH 6.0, and concentrated to avolume of 0.5 ml as described above. The ELISA method described belowwas used to measure the antibody concentration in each sample.

Stable Transfections:

Sp2/0 cells were stably transfected with the appropriate light chainplasmid and the appropriate wild-type or one of the various mutatedheavy chain plasmids containing a single or double amino acidsubstitution at position 250 or 428. Approximately 1×10⁷ cells werewashed with 10 ml of PBS, and resuspended in 1 ml of PBS. Approximately25-30 μg of light chain plasmid and 50-60 μg of heavy chain plasmid werelinearized with FspI, and added to the cells. Cells and DNA were mixedgently, and transferred to a Gene Pulser Cuvette (Bio-Rad® Laboratories,Hercules, Calif.) on ice. The cells were electroporated using a GenePulser II (Bio-Rad® Laboratories) set at 0.360 kV, 25 μF, and returnedto ice for 10-20 minutes. The cells were diluted in 40 ml of DMEM, 10%FBS, 2 mM L-glutamine, and plated in four 96-well plates at 100 μl/well.After 48 hours, 100 μl/well of 2× mycophenolic acid (MPA) selectionmedium (DMEM, 10% FBS, 1×HT Media Supplement Hybri-Max® (Sigma®, St.Louis, Mo.), 300 μg/ml xanthine (Sigma®), 2 μg/ml mycophenolic acid(Life Technologies®), and 2 mM L-glutamine) was added. Supernatants fromwells apparently containing single colonies were screened by ELISA after10-14 days. The highest antibody-producing clones were chosen forexpansion and adaptation to HSFM (Life Technologies®). Adapted cloneswere expanded to roller bottles in 450 ml of HSFM, gassed with 5% CO₂ inair, supplemented after 2 days with 50 ml of Protein Free Feed Medium-2(PFFM-2) (Sauer et al., Biotechnol. Bioeng. 67:585-597 (2000)), andgrown to exhaustion.

Some of the highest antibody-producing clones were also adapted toProtein-Free Basal Medium-1 (PFBM-1) (Protein Design Labs™, Inc.),expanded to 10 L spinner flasks, supplemented after 2 days with 1/10volume of PFFM-2, and grown to exhaustion.

ELISA:

To quantitate the amount of OST577 or Hu1D10 antibody present in culturesupernatants, an ELISA was performed. Immulon™ 4 plates (DYNEX®Technologies, Inc., Chantilly, Va.) were coated overnight at 4° C. with1.0 μg/ml of goat F(ab′)₂ anti-human IgG gamma chain antibody (BioSourceInternational, Camarillo, Calif.) or AffiniPure™ goat anti-human IgG Fcγfragment specific antibody (Jackson ImmunoResearch Laboratories, Inc.,West Grove, Pa.) in 100 μl/well of 0.2 M carbonate-bicarbonate buffer,pH 9.4. The next day, the plates were washed with ELISA Wash Buffer(EWB) (PBS, 0.1% Tween 20) and blocked with 300 μl/well of SuperBlock®Blocking Buffer in TBS (Pierce Chemical Company, Rockford, Ill.) for20-30 minutes at room temperature. The plates were washed with EWB, andappropriately diluted test samples were added to each well. PurifiedOST577 or Hu1D10 antibodies, as appropriate, were serially dilutedtwofold in 100 μl/well of ELISA Buffer (EB) (PBS, 1% bovine serumalbumin, 0.1% Tween 20) starting at 0.2 μg/ml, and used as standards.Culture supernatants were initially diluted 1:10 in 100 μl/well of EB,then serially diluted twofold in 100 μl/well of EB. The plates wereincubated for 1-2 hours at room temperature, then washed with EWB, and100 μl/well of goat anti-human lambda light chain HRP-conjugatedantibody (BioSource International, or Southern Biotechnology Associates,Inc., Birmingham, Ala.) or goat anti-human kappa light chainHRP-conjugated antibody (Southern Biotechnology Associates, Inc.), asappropriate, was added at 1.0 μg/ml in EB. After incubation for 1 hourat room temperature, the plates were washed with EWB, followed byaddition of 100 μl/well of ABTS Peroxidase Substrate/Peroxidase SolutionB (Kirkegaard & Perry Laboratories, Gaithersburg, Md.). The reaction wasstopped with 100 μl/well of 2% oxalic acid, and the absorbance at 415 nmwas measured using a VERSAmax™ microtitre plate reader (MolecularDevices® Corporation, Sunnyvale, Calif.).

Antibody Purification:

Culture supernatants from transient transfections were harvested bycentrifugation, and sterile filtered. The pH of the filteredsupernatants was adjusted by addition of 1/50 volume of 1 M sodiumcitrate, pH 7.0. Supernatants were run over a 1 ml HiTrap® Protein A HPcolumn (Amersham Biosciences™ Corporation, Piscataway, N.J.) that waspre-equilibrated with 20 mM sodium citrate, 150 mM NaCl, pH 7.0. Thecolumn was washed with the same buffer, and bound antibody was elutedwith 20 mM sodium citrate, pH 3.5. After neutralization by addition of1/50 volume of 1.5 M sodium citrate, pH 6.5, the pooled antibodyfractions were run over a 5 ml HiTrap® Desalting column (AmershamBiosciences™ Corporation) that was pre-equilibrated with 20 mM sodiumcitrate, 120 mM NaCl, pH 6.0. The flow-through was collected andfractions with OD₂₈₀>0.1 were pooled and concentrated to ˜0.5-1.0 mg/mlusing 2 ml Vivaspin® concentrators (50,000 dalton MWCO) (Vivascience®AG). Samples were then filter sterilized using 0.2 μm Millex®-GVmicrofilters (Millipore® Corporation). The concentrations of thepurified antibodies were determined by UV spectroscopy by measuring theabsorbance at 280 nm (1 mg/ml=1.4 A₂₈₀).

For small-scale purification of antibody from stable transfections,culture supernatants were harvested by centrifugation, and sterilefiltered. Supernatants were run over a 5 ml POROS® 50A Protein A column(Applied Biosystems®) that was pre-equilibrated with PBS, pH 7.4. Thecolumn was washed with the same buffer, and bound antibody was elutedwith 0.1 M glycine, 0.1 M NaCl, pH 3.0. After neutralization by additionof 1/20 volume of 1 M Tris base, pooled fractions were buffer exchangedinto PBS, pH 7.4, using a PD-10 desalting column (Amersham Biosciences™Corporation) or by dialysis. Samples were then filter sterilized using0.2 μm Millex®-GV microfilters (Millipore® Corporation). Theconcentrations of the purified antibodies were determined by UVspectroscopy by measuring the absorbance at 280 nm (1 mg/ml=1.4 A₂₈₀).

For large-scale purification of antibody from stable transfectants, thecell culture harvest was clarified by dead-end filtration usingSartorius® filtration capsules (Sartorius® AG, Goettingen, Germany). Theclarified harvest was concentrated from approximately 10 L to 750 mlusing a Pellicon® 2 cassette (30,000 dalton MWCO) (Millipore®Corporation), then purified by protein A affinity chromatography on arProtein A Sepharose FF column (Amersham Biosciences™ Corporation) usingthe citrate buffer system described above. The protein A eluate wasconcentrated in an Amicon® stir cell apparatus using a YM30 membrane(Millipore® Corporation), then the sample was buffer exchanged into 20mM sodium citrate, 120 mM NaCl, pH 6.0, using a Superdex™ 200 column(Amersham Biosciences™ Corporation). The pH and osmolality weremeasured, and the concentrations of the purified antibodies weredetermined by UV spectroscopy by measuring the absorbance at 280 nm (1mg/ml=1.4 A₂₈₀).

SDS-PAGE:

Five μg samples of purified antibodies were run under reducing ornon-reducing conditions on NuPAGE® Novex 4-12% Bis-Tris gels(Invitrogen™) and stained using the SimplyBlue™ SafeStain Kit(Invitrogen™) following the manufacturer's recommendations.

Results:

The IgG2M3 Fc and IgG1 Fc mutants were expressed as anti-HBV antibodies,comprising the light and heavy chain variable regions of OST577 (Ehrlichet al., op. cit.), the light chain constant region of human lambda-2(Hieter et al. (1981), op. cit.), and the heavy chain constant regionsof human gamma-2 mutant 3 (IgG2M3) (Cole et al, op. cit.) and IgG1(Ellison et al., op. cit.), respectively. The IgG2M3 variant containstwo amino acid substitutions in the C_(H)2 region (V234A and G237A), andshows extremely low residual binding to human Fcγ receptors (Cole etal., op. cit.). The IgG2M3 Fc and IgG1 Fc mutants were also expressed asanti-HLA-DR β chain allele antibodies, comprising the light and heavychain variable regions of Hu1D10 (Kostelny et al. (2001), op. cit.), thelight chain constant region of human kappa (Hieter et al. (1980), op.cit.), and the heavy chain constant regions of human IgG2M3 (Cole et al,op. cit.) and IgG1 (Ellison et al., op. cit.), respectively. Asdescribed above, the appropriate wild-type or mutant heavy chainexpression vector was transiently co-transfected with the appropriatelight chain expression vector into 293-H cells for expression of OST577or Hu1D10 monoclonal antibodies. ELISA analysis of culture supernatantsharvested 5-7 days after transient transfection showed that antibodyexpression levels were typically 5-50 μg/ml in 25 ml of supernatant.OST577 or Hu1D10 antibodies were purified by protein A affinitychromatography for a final yield of approximately 100-1000 μg. Stableexpression of OST577 or Hu1D10 antibodies in Sp2/0 cells typicallyresulted in expression levels of 5-50 μg/ml as determined by ELISA.Yields of approximately 50-80% of the antibody present in culturesupernatants were obtained by small-scale protein A affinitychromatography.

Purified antibodies were characterized by SDS polyacrylamide gelelectrophoresis (SDS-PAGE) under non-reducing and reducing conditions.SDS-PAGE analysis under non-reducing conditions indicated that thepurified antibodies had a molecular weight of about 150-160 kD (data notshown); analysis under reducing conditions indicated that the purifiedantibodies were comprised of a heavy chain with a molecular weight ofabout 50 kD and a light chain with a molecular weight of about 25 kD(see FIGS. 10A and 10B). SDS-PAGE analysis of antibodies purified fromstable Sp2/0 transfectants gave results similar to those observed withantibodies purified from transient 293-H transfections.

Example 6

This example describes the competitive binding analysis of mutant IgG2M3and IgG1 antibodies.

Cell Culture:

Mouse myeloma cell line NS0 (European Collection of Animal CellCultures, Salisbury, Wiltshire, UK) was maintained in DMEM containing10% FBS. NS0 transfectants expressing recombinant, GPI-linked human orrhesus FcRn on the surface were maintained in mycophenolic acid (MPA)selection medium (DMEM, 10% FBS, 1×HT Media Supplement Hybri-Max®(Sigma®), 250 μg/ml xanthine (Sigma®), 1 μg/ml mycophenolic acid (LifeTechnologies®), and 2 mM L-glutamine) or 2×MPA selection medium.

Human FcRn Cell Line:

NS0 cells were stably transfected with pDL208. Approximately 1×10⁷ cellswere washed once and resuspended in 1 ml of plain DMEM, transferred to aGene Pulser™ Cuvette (Bio-Rad® Laboratories), and incubated on ice for10 minutes. Forty μg of plasmid pDL208 was linearized with FspI andgently mixed with the cells on ice, then the cells were electroporatedby pulsing twice using a Gene Pulser™ II (Bio-Rad® Laboratories) set at1.5 kV, 3 μF, and returned to ice for 10 minutes. The cells were dilutedin 20 ml of DMEM, 10% FBS, and plated in two 96-well plates at 100μl/well. The medium was replaced after 48 hours with MPA selectionmedium. Mycophenolic acid-resistant NS0 transfectants from wellsapparently containing single colonies were expanded in MPA selectionmedium and screened after about 3 weeks by FACS™. Approximately 1.5×10⁵cells/test were incubated in 100 μl of FACS Staining Buffer (FSB) (PBS,1% FBS, 0.1% NaN₃) containing 10 μg/ml of biotinylated mouse anti-humanβ-microglobulin antibody (Chromaprobe, Inc., Aptos, Calif.) for 1 houron ice. The cells were washed once with 4 ml of FSB, then incubated in25 μl of FSB containing 20 μg/ml of streptavidin-FITC conjugate(Southern Biotechnology Associates, Inc.) for 30 minutes on ice in thedark. The cells were washed once with 4 ml of FSB, and resuspended in 1%formaldehyde. Samples were analyzed for antibody binding to human β2musing a FACScan flow cytometer (BD® Biosciences, San Jose, Calif.).Several clones with the highest apparent staining were subcloned using aFACStar cell sorter (BD® Biosciences), expanded in DMEM, 10% FBS, 2 mML-glutamine, and retested by FACS™ as described above. One subclone,designated NS0 HuFcRn (memb), clone 7-3, was used in subsequent bindingassays.

Rhesus FcRn Cell Line:

NS0 cells were stably transfected with pDL410. Approximately 6×10⁵ cellswere transfected by electroporation as described above. Transfectantswere identified by FACS™ as described above by staining with 100 ng/testof a mouse anti-human FcRn alpha chain antibody (Protein Design Labs™,Inc.) that cross-reacts with rhesus FcRn alpha chain, and detecting withgoat anti-mouse kappa FITC-conjugated antibody (Southern BiotechnologyAssociates, Inc.). A cell line designated NS0 RhFcRn, clone R-3, wasused in subsequent binding assays.

Single Point Competitive Binding Assay:

Concentrated OST577-IgG2M3 supernatants were tested in a single-pointcompetitive binding assay for binding to human FcRn on cell line NS0HuFcRn (memb), clone 7-3. Approximately 2×10⁵ cells/test were washedonce in FACS Binding Buffer (FBB) (PBS containing 0.5% BSA, 0.1% NaN₃),pH 8.0, once in FBB, pH 6.0, and resuspended in 120 μl of pre-mixedbiotinylated OST577-IgG2M3 antibody (8.3 μg/ml) and concentratedsupernatant (containing 8.3 μg/ml of competitor antibody) in FBB, pH6.0. The cells were incubated for 1 hour on ice, washed twice in FBB, pH6.0, and resuspended in 25 μl of streptavidin-RPE conjugate (BioSourceInternational) diluted to 2.5 μg/ml in FBB, pH 6.0. After incubation for30 minutes on ice in the dark, the cells were washed twice in FBB, pH6.0, and resuspended in 1% formaldehyde. Samples were analyzed forantibody binding to FcRn by FACS™ using a FACSCalibur flow cytometer(BD® Biosciences). Mean channel fluorescence (MCF) of each mutant wascompared to that of the wild-type antibody and plotted using Excel(Microsoft® Corporation, Redmond, Wash.).

Competitive Binding Assays:

A dilution series of each purified OST577-IgG2M3 antibody was competedagainst biotinylated HuEP5C7-IgG2M3 antibody (He et al., J. Immunol.160:1029-1035 (1998)) for binding to human FcRn on cell line NS0 HuFcRn(memb), clone 7-3. For initial screening experiments, approximately2×10⁵ cells/test were washed once in FSB, pH 6.0, and resuspended in 100μl of pre-mixed biotinylated HuEP5C7-IgG2M3 antibody (10 μg/ml) andOST577-IgG2M3 competitor antibody (twofold serial dilutions from 208μg/ml to 0.102 μg/ml) in FSB, pH 6.0. The cells were incubated with theantibody mixture for 1 hour on ice, washed twice in FSB, pH 6.0, andresuspended in 25 μl of streptavidin-RPE conjugate (BioSourceInternational) diluted to 2.5 μg/ml in FSB, pH 6.0. After incubation for30 minutes on ice in the dark, the cells were washed twice in FSB, pH6.0, and resuspended in 1% formaldehyde. Samples were analyzed forantibody binding to FcRn by FACS™ using a FACScan flow cytometer (BD®Biosciences). Mean channel fluorescence (MCF) was plotted againstcompetitor concentration, and IC50 values were calculated using GraphPadPrism® (GraphPad™ Software, Inc., San Diego, Calif.). For consistency,the IC50 values shown in the Tables are based on the final competitorconcentrations.

Subsequent competitive binding experiments were done as described above,except the cells were washed once in FBB, pH 8.0, and once in FBB, pH6.0, then resuspended in 100 μl of pre-mixed biotinylated HuEP5C7-IgG2M3antibody (10 μg/ml) and OST577-IgG2M3 competitor antibody (twofoldserial dilutions from 208 μg/ml to 0.102 μg/ml) in FBB, pH 6.0. Allsubsequent incubations and washes were done using FBB, pH 6.0, asdescribed above. One group of experiments was done in 120 μl ofpre-mixed biotinylated OST577-IgG2M3 antibody (8.3 μg/ml) andOST577-IgG2M3 competitor antibody (twofold serial dilutions from 208μg/ml to 0.102 μg/ml) in FBB, pH 6.0, as described above. Another groupof experiments was done in 200 μl of pre-mixed biotinylated OST577-IgG1antibody (5.0 μg/ml) and OST577-IgG1 competitor antibody (twofold serialdilutions starting from 125 μg/ml, or threefold serial dilutionsstarting from 250 μg/ml) in FBB, pH 6.0, as described above. A furthergroup of experiments was done in 200 μl of pre-mixed biotinylatedOST577-IgG1 antibody (5.0 μg/ml) and OST577-IgG1 competitor antibody(threefold serial dilutions starting from 750 μg/ml) in FBB, pH 6.0, asdescribed above.

A dilution series of each purified OST577-IgG2M3 antibody was competedagainst biotinylated OST577-IgG2M3 antibody for binding to rhesus FcRnon cell line NS0 RhFcRn, clone R-3. In one group of experiments,approximately 2×10⁵ cells/test were washed once in FBB, pH 8.0, and oncein FBB, pH 6.0, then resuspended in 120 μl of pre-mixed biotinylatedOST577-IgG2M3 antibody (8.3 μg/ml) and OST577-IgG2M3 competitor antibody(twofold serial dilutions from 208 μg/ml to 0.102 μg/ml) in FBB, pH 6.0.The cells were incubated with the antibody mixture for 1 hour on ice,washed twice in FBB, pH 6.0, and resuspended in 25 μl ofstreptavidin-RPE conjugate (BioSource International) diluted to 2.5μg/ml in FBB, pH 6.0. After incubation for 30 minutes on ice in thedark, the cells were washed twice in FBB, pH 6.0, and resuspended in 1%formaldehyde. Samples were analyzed for antibody binding to FcRn byFACS™ using a FACSCalibur flow cytometer (BD® Biosciences). Anothergroup of experiments was done in 200 μl of pre-mixed biotinylatedOST577-IgG1 antibody (5.0 μg/ml) and OST577-IgG1 competitor antibody(threefold serial dilutions starting from 500 μg/ml) in FBB, pH 6.0, asdescribed above.

Results:

The relative binding of wild-type OST577-IgG2M3 or OST577-IgG1antibodies and their various mutants to FcRn was determined using atransfected NS0 cell line stably expressing human FcRn on its surface.As described above, the concentrated supernatants were tested forbinding to human FcRn according to the single point competitive bindingassay and the purified antibodies were tested for FcRn binding in thecompetitive binding assays. Increasing concentrations of unlabeledcompetitor antibodies were incubated with cells in the presence of asub-saturating concentration of labeled IgG2M3 or IgG1 antibody in FSBor FBB, pH 6.0.

The results of typical experiments with concentrated OST577-IgG2M3supernatants are shown in FIGS. 11A, 11B, and 11C. As shown in FIG. 11A,some of the mutants at position 250 (e.g., T250E, T250Q) were strongercompetitors than the wild-type, suggesting that these mutants haveincreased binding to human FcRn as compared to the wild-type antibody.Other mutants at this position (e.g., T250D, T250F, T250K, T250N, T250P,T250R, T250W, T250Y) were weaker competitors than the wild-type,suggesting that these mutants have reduced binding to human FcRn ascompared to the wild-type antibody. As shown in FIG. 11B, none of themutants at position 314 was a stronger competitor than the wild-type,suggesting that none of these mutants has increased binding to humanFcRn as compared to the wild-type antibody. Most of the mutants at thisposition (e.g., L314A, L314C, L314D, L314E, L314F, L314G, L314H, L314K,L314M, L314N, L314P, L314Q, L314R, L314S, L314T, L314V, L314W, L314Y)were weaker competitors than the wild-type, suggesting that thesemutants have reduced binding to human FcRn as compared to the wild-typeantibody. As shown in FIG. 11C, some of the mutants at position 428(e.g., M428F, M428L) were stronger competitors than the wild-type,suggesting that these mutants have increased binding to human FcRn ascompared to the wild-type antibody. Other mutants at this position(e.g., M428A, M428C, M428D, M428E, M428G, M428H, M428K, M428N, M428P,M428Q, M428R, M428S, M428T, M428V, M428Y) were weaker competitors thanthe wild-type, suggesting that these mutants have reduced binding tohuman FcRn as compared to the wild-type antibody.

Table 2 summarizes the IC50 values (the amount of competitor antibodynecessary to inhibit binding of the labeled antibody to FcRn by 50%) ofthe purified wild-type OST577-IgG2M3 antibody and some of its mutantsfor binding to human FcRn. Relative binding values were calculated asthe ratio of the IC50 value of the wild-type OST577-IgG2M3 antibody tothat of each of the mutants. At amino acid position 314, none of thepurified mutants showed an increase in binding to human FcRn relative tothe wild-type antibody. In fact, all four of the purified mutants atposition 314 showed reduced binding relative to the wild-type antibody.However, at amino acid position 250, one of the mutants (T250E) showedapproximately 3-fold better binding to human FcRn than the wild-typeantibody. Several mutants at position 250 showed slightly reducedbinding relative to the wild-type antibody, and one mutant (T250D)showed substantially reduced binding to human FcRn relative to thewild-type antibody. At amino acid position 428, one of the mutants(M428F) also showed approximately 3-fold better binding to human FcRnthan the wild-type antibody, while another mutant (M428G) showedsubstantially reduced binding to human FcRn relative to the wild-typeantibody.

Since two amino acid substitutions at two different positions wereidentified, namely T250E, T250Q, M428F, and M428L, each showing anincrease in binding to human FcRn, the double mutants T250E/M428F,T250Q/M428F, and T250Q/M428L were constructed, transiently transfectedinto 293-H cells, purified, and tested for binding to human FcRn. Asdescribed above, increasing concentrations of unlabeled competitorantibodies were incubated with cells expressing human FcRn in thepresence of a sub-saturating concentration of labeled HuEP5C7-IgG2M3 orOST577-IgG2M3 antibody in FBB, pH 6.0.

As shown in FIG. 12A, the double mutant (T250E/M428F) showed betterbinding to human FcRn than either of the single mutants (T250E or M428F)and showed approximately 15-fold better binding to human FcRn than thewild-type antibody. As shown in FIG. 12B, the double mutant(T250Q/M428L) showed better binding to human FcRn than either of thesingle mutants (T250Q or M428L) or the double mutant (T250Q/M428F), andshowed approximately 28-fold better binding to human FcRn than thewild-type antibody. As summarized in Table 3, in one group ofexperiments, the IC50 for the wild-type OST577-IgG2M3 antibody is ˜9μg/ml, whereas the IC50 for each of the single mutants (T250E and M428F)is ˜3 μg/ml, and the IC50 for the double mutant (T250E/M428F) is lessthan 1 μg/ml. As summarized in Table 4, in another group of experiments,the IC50 for the wild-type OST577-IgG2M3 antibody is ˜12 μg/ml, whereasthe IC50 for each of the single mutants (T250Q and M428L) and the doublemutant (T250Q/M428F) is ˜2-4 μg/ml, and the IC50 for the double mutant(T250Q/M428L) is less than 1 μg/ml.

Wild-type OST577-IgG1, the single mutants T250E, T250Q, M428F, M428L,and the double mutants T250E/M428F and T250Q/M428L were also created. Assummarized in Table 5, in one group of experiments, the IC50 for thewild-type OST577-IgG1 antibody is ˜14 μg/ml, whereas the IC50 for eachof the single mutants (T250E and M428F) is ˜3-5 μg/ml, and the IC50 forthe double mutant (T250E/M428F) is less than 1 μg/ml. As summarized inTable 6, in another group of experiments, the IC50 for the wild-typeOST577-IgG1 antibody is ˜10 μg/ml, whereas the IC50 for the singlemutant (T250Q) is ˜3 μg/ml, and the IC50 for the single mutant (M428L)and the double mutant (T250Q/M428L) is less than 1 μg/ml.

The binding of OST577-IgG2M3 and some of its mutants to rhesus FcRn weretested in competitive binding experiments. As summarized in Table 7, theIC50 for the wild-type OST577-IgG2M3 antibody is ˜15 μg/ml, whereas theIC50 for each of the single mutants (T250Q and M428L) and the doublemutant (T250Q/M428F) is ˜2-4 μg/ml, and the IC50 for the double mutant(T250Q/M428L) is less than 1 μg/ml. The binding of OST577-IgG1 and someof its mutants to rhesus FcRn were also tested in competitive bindingexperiments. As summarized in Table 8, the IC50 for the wild-typeOST577-IgG1 antibody is ˜9 μg/ml, whereas the IC50 for the single mutant(T250Q) is ˜3 μg/ml, and the IC50 for the single mutant (M428L) and thedouble mutant (T250Q/M428L) is less than 1 μg/ml.

TABLE 2 Name^(a) (IgG2M3) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 6 10.7 ± 4.6 1.0 L314W 2 20.2 ± 2.0 0.53 L314Q 2 32.0 ± 2.30.33 L314A 2 68.7 ± 2.1 0.16 L314R 2 102 ± 4  0.11 T250E 1 3.82 2.8T250S 2 12.0 ± 0.9 0.89 T250A 2 13.3 ± 0.5 0.80 T250V 3 19.0 ± 1.8 0.56T250D 1 >210 <0.050 M428F 2 3.40 ± 0.53 3.1 M428G 1 147 0.072 ^(a)Foreach mutant, the first letter indicates the wild-type amino acid, thenumber indicates the position according to the EU index (Kabat et al.,op. cit.), and the second letter indicates the mutant amino acid. ^(b)nindicates the number of independent assays. ^(c)IC50 values (±S.D.) areexpressed in μg/ml (based on final competitor concentrations) and werecalculated from competitive binding assays versus biotinylatedHuEP5C7-IgG2M3 in FSB, pH 6.0, as described in Example 6. ^(d)Relativebinding to human FcRn was calculated as the ratio of the IC50 value ofthe wild-type OST577-IgG2M3 to that of each of the mutants.

TABLE 3 Name^(a) (IgG2M3) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 3 9.40 ± 2.87 1.0 T250E 3 2.71 ± 1.16 3.5 M428F 3 2.97 ± 0.303.2 T250E/M428F 2 0.639 ± 0.094 15 ^(a)For each mutant, the first letterindicates the wild-type amino acid, the number indicates the positionaccording to the EU index (Kabat et al., op. cit.), and the secondletter indicates the mutant amino acid. ^(b)n indicates the number ofindependent assays. ^(c)IC50 values (±S.D.) are expressed in μg/ml(based on final competitor concentrations) and were calculated fromcompetitive binding assays versus biotinylated HuEP5C7-IgG2M3 in FBB, pH6.0, as described in Example 6. ^(d)Relative binding to human FcRn wascalculated as the ratio of the IC50 value of the wild-type OST577-IgG2M3to that of each of the mutants.

TABLE 4 Name^(a) (IgG2M3) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 3 11.9 ± 2.5  1.0 T250Q 3 4.20 ± 1.02 2.8 M428L 3 1.79 ± 0.696.7 T250Q/M428F 3 1.50 ± 0.31 8.0 T250Q/M428L 3 0.430 ± 0.084 28 ^(a)Foreach mutant, the first letter indicates the wild-type amino acid, thenumber indicates the position according to the EU index (Kabat et al.,op. cit.), and the second letter indicates the mutant amino acid. ^(b)nindicates the number of independent assays. ^(c)IC50 values (±S.D.) areexpressed in μg/ml (based on final competitor concentrations) and werecalculated from competitive binding assays versus biotinylatedOST577-IgG2M3 in FBB, pH 6.0, as described in Example 6. ^(d)Relativebinding to human FcRn was calculated as the ratio of the IC50 value ofthe wild-type OST577-IgG2M3 to that of each of the mutants.

TABLE 5 Name^(a) (IgG1) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 6 13.9 ± 4.2  1.0 T250E 3 5.26 ± 0.87 2.6 M428F 5 3.44 ± 1.224.0 T250E/M428F 4 0.990 ± 0.786 14 ^(a)For each mutant, the first letterindicates the wild-type amino acid, the number indicates the positionaccording to the EU index (Kabat et al., op. cit.), and the secondletter indicates the mutant amino acid. ^(b)n indicates the number ofindependent assays. ^(c)IC50 values (±S.D.) are expressed in μg/ml(based on final competitor concentrations) and were calculated fromcompetitive binding assays versus biotinylated OST577-IgG1 in FBB, pH6.0, as described in Example 6. ^(d)Relative binding to human FcRn wascalculated as the ratio of the IC50 value of the wild-type OST577-IgG1to that of each of the mutants.

TABLE 6 Name^(a) (IgG1) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 5 10.3 ± 2.8  1.0 T250Q 5 3.14 ± 0.86 3.3 M428L 5 0.896 ±0.304 11 T250Q/M428L 5 0.351 ± 0.144 29 ^(a)For each mutant, the firstletter indicates the wild-type amino acid, the number indicates theposition according to the EU index (Kabat et al., op. cit.), and thesecond letter indicates the mutant amino acid. ^(b)n indicates thenumber of independent assays. ^(c)IC50 values (±S.D.) are expressed inμg/ml (based on final competitor concentrations) and were calculatedfrom competitive binding assays versus biotinylated OST577-IgG1 in FBB,pH 6.0, as described in Example 6. ^(d)Relative binding to human FcRnwas calculated as the ratio of the IC50 value of the wild-typeOST577-IgG1 to that of each of the mutants.

TABLE 7 Name^(a) (IgG2M3) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 3 14.8 ± 2.7  1.0 T250Q 3 4.05 ± 0.24 3.6 M428L 3 1.92 ± 0.467.7 T250Q/M428F 3 1.77 ± 0.60 8.4 T250Q/M428L 3 0.554 ± 0.052 27 ^(a)Foreach mutant, the first letter indicates the wild-type amino acid, thenumber indicates the position according to the EU index (Kabat et al.,op. cit.), and the second letter indicates the mutant amino acid. ^(b)nindicates the number of independent assays. ^(c)IC50 values (±S.D.) areexpressed in μg/ml (based on final competitor concentrations) and werecalculated from competitive binding assays versus biotinylatedOST577-IgG2M3 in FBB, pH 6.0, as described in Example 6. ^(d)Relativebinding to rhesus FcRn was calculated as the ratio of the IC50 value ofthe wild-type OST577-IgG2M3 to that of each of the mutants.

TABLE 8 Name^(a) (IgG1) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 3 8.86 ± 0.52 1.0 T250Q 3 2.97 ± 0.59 3.0 M428L 3 0.629 ±0.060 14 T250Q/M428L 3 0.236 ± 0.013 37 ^(a)For each mutant, the firstletter indicates the wild-type amino acid, the number indicates theposition according to the EU index (Kabat et al., op. cit.), and thesecond letter indicates the mutant amino acid. ^(b)n indicates thenumber of independent assays. ^(c)IC50 values (±S.D.) are expressed inμg/ml (based on final competitor concentrations) and were calculatedfrom competitive binding assays versus biotinylated OST577-IgG1 in FBB,pH 6.0, as described in Example 6. ^(d)Relative binding to rhesus FcRnwas calculated as the ratio of the IC50 value of the wild-typeOST577-IgG1 to that of each of the mutants.

Example 7

This example describes confirmation of the properties of the IgG2M3 andIgG1 mutants in FcRn binding.

Direct Binding Assay:

Purified OST577-IgG2M3 antibodies were tested for binding to human FcRnon cell line NS0 HuFcRn (memb), clone 7-3, or to untransfected NS0 cellsin FBB at pH 6.0. Approximately 2×10⁵ cells/test were washed once inFBB, pH 8.0, and once in FBB, pH 6.0, then resuspended in 100 μl ofantibody at a concentration of 11 μg/ml in FBB, pH 6.0. The cells wereincubated with antibody for 1 hour on ice, washed twice in FBB, pH 6.0,and resuspended in 25 μl of goat anti-human IgG RPE-conjugated antibody(Southern Biotechnology Associates, Inc.) diluted to 5 μg/ml in FBB, pH6.0. After incubation for 30 minutes on ice in the dark, the cells werewashed twice in FBB, pH 6.0, and resuspended in 1% formaldehyde. Sampleswere analyzed for antibody binding to FcRn by FACS™ using a FACScan flowcytometer (BD® Biosciences). Mean channel fluorescence (MCF) of eachmutant was plotted using Excel (Microsoft® Corporation).

Competitive Binding Assay at 37° C.:

A dilution series of each purified OST577-IgG2M3 antibody was competedagainst biotinylated OST577-IgG2M3 antibody for binding to human FcRn oncell line NS0 HuFcRn (memb), clone 7-3 at 37° C. Approximately 2×10⁵cells/test were washed once in FBB, pH 8.0, and once in FBB, pH 6.0,then resuspended in 100 μl of pre-mixed biotinylated OST577-IgG2M3antibody (10 μg/ml) and OST577-IgG2M3 competitor antibody (twofoldserial dilutions, from 208 μg/ml to 0.102 μg/ml) in FBB, pH 6.0. Thecells were incubated with the antibody mixture for 1 hour at 37° C.,washed twice in FBB, pH 6.0, and resuspended in 25 μl ofstreptavidin-RPE conjugate (BioSource International) diluted to 2.5μg/ml in FBB, pH 6.0. After incubation for 30 minutes in the dark, thecells were washed twice in FBB, pH 6.0, and resuspended in 1%formaldehyde. Samples were analyzed for antibody binding to FcRn byFACS™ using a FACScan flow cytometer (BD® Biosciences). Mean channelfluorescence (MCF) was plotted against competitor concentration, andIC50 values were calculated using GraphPad Prism® (GraphPad™ Software).

pH-Dependent Binding and Release Assay:

Purified OST577-IgG2M3 and OST577-IgG1 mutant antibodies were comparedto the respective wild-type antibodies for binding to human FcRn andthen released at various pH values in single-point binding and releaseassays using cell line NS0 HuFcRn (memb), clone 7-3. Approximately 2×10⁵cells/test were washed once in FBB, pH 8.0, and once in FBB, pH 6.0,then resuspended in 100 μl of purified antibody (10 μg/ml) in FBB, pH6.0. The cells were incubated for 1 hour on ice, washed twice in FBB, pH6.0, 6.5, 7.0, 7.5, or 8.0, and resuspended in 25/t of goat F(ab′)₂anti-human IgG FITC-conjugated antibody (Southern BiotechnologyAssociates, Inc.) diluted to 1.25 μg/ml in FBB of the appropriate pH.After incubation for 30 minutes on ice in the dark, the cells werewashed twice in FBB of the appropriate pH, and resuspended in 1%formaldehyde. Samples were analyzed for antibody binding to FcRn byFACS™ using a FACSCalibur flow cytometer (BD® Biosciences). Mean channelfluorescence (MCF) of each mutant was plotted using Excel (Microsoft®Corporation).

Purified OST577-IgG2M3 and OST577-IgG1 mutant antibodies were comparedto the respective wild-type antibodies for binding to rhesus FcRn andthen released at various pH values in single-point binding and releaseassays using cell line NS0 RhFcRn, clone R-3, as described above.

Results:

The binding properties of wild-type OST577-IgG2M3 or OST577-IgG1antibodies and their various mutants to human FcRn were confirmed usinga transfected NS0 cell line stably expressing human FcRn on its surface.To confirm that binding of the mutant antibodies was specific for humanFcRn on the transfected NS0 cell line, rather than occurring via someother receptor or by an unknown mechanism, the antibodies were testedfor binding to untransfected NS0 cells versus a transfected NS0 cellline stably expressing human FcRn. As described above, the cells wereincubated with a sub-saturating concentration of antibody in FBB, pH6.0, and binding was analyzed by FACS™. As shown in FIG. 13, the resultsindicated that there was no apparent binding to the parent NS0 cellline, suggesting that the antibodies bind specifically to thetransfected cells via human FcRn.

To confirm that each of the mutants generated in the present inventionbehaved in a physiologically relevant manner, the effects of temperatureand pH on binding to human FcRn were examined more closely. Because theinitial competitive binding assays were performed at 4° C., theexperiments were repeated at the more physiologically relevanttemperature of 37° C. to show that the mutants were still active at thistemperature. As described above, increasing concentrations of unlabeledcompetitor antibodies were incubated with cells expressing human FcRn inthe presence of a sub-saturating concentration of labeled OST577-IgG2M3antibody at 37° C. in FBB, pH 6.0. As shown in FIG. 14, the resultsindicated that the antibodies maintained their relative pattern ofbinding to human FcRn at 37° C.

The binding of IgG to FcRn is known to be pH-dependent: IgG bindsstrongly to FcRn at pH 6.0 but weakly at pH 8.0. In order to engineermutant antibodies with longer serum half-lives, it is desirable toincrease binding to FcRn at pH 6.0, while retaining pH-dependent releasefrom FcRn at pH 8.0. To confirm that binding was pH-dependent, theantibodies were tested for binding to a transfected NS0 cell line stablyexpressing human FcRn and then released at pH values ranging from pH 6.0to pH 8.0. As described above, the cells were incubated with asub-saturating concentration of antibody in FBB, pH 6.0, washed withFBB, pH 6.0, 6.5, 7.0, 7.5, or 8.0, and binding was analyzed by FACS™.As shown in FIG. 15A, the results indicated that the modifiedOST577-IgG2M3 antibodies having the T250E, T250Q, M428F, M428L,T250E/M428F, T250Q/M428F, or T250Q/M428L mutations all showed strongbinding to human FcRn at pH 6.0, with diminishing binding as the pHvalues increased to pH 8.0. As shown in FIG. 15B, the results indicatedthat the modified OST577-IgG1 antibodies having the T250E, M428F, orT250E/M428F mutations all showed strong binding to human FcRn at pH 6.0,with diminishing binding as the pH values increased to pH 8.0. TheOST577-IgG1 antibody having the T250D mutation showed weaker binding(compared to wild-type) to human FcRn at pH 6.0, with diminishingbinding as the pH values increased to pH 8.0. As shown in FIG. 15C, theresults indicated that the modified OST577-IgG1 antibodies having theT250Q, M428L, or T250Q/M428L mutations all showed strong binding tohuman FcRn at pH 6.0, with diminishing binding as the pH valuesincreased to pH 8.0. These results indicated that the binding of theantibodies to human FcRn was indeed pH-dependent.

Similarly, the antibodies were tested for binding to a transfected NS0cell line stably expressing rhesus FcRn and then released at pH valuesranging from pH 6.0 to pH 8.0. As shown in FIG. 15D, the resultsindicated that the modified OST577-IgG2M3 antibodies having the T250E,T250Q, M428F, M428L, T250E/M428F, T250Q/M428F, or T250Q/M428L mutationsall showed strong binding to rhesus FcRn at pH 6.0, with diminishingbinding as the pH values increased to pH 8.0. As shown in FIG. 15E, theresults indicated that the modified OST577-IgG1 antibodies having theT250Q, M428L, or T250Q/M428L mutations all showed strong binding torhesus FcRn at pH 6.0, with diminishing binding as the pH valuesincreased to pH 8.0. These results indicated that the binding of theantibodies to rhesus FcRn was also pH-dependent.

Example 8

This example describes confirmation of additional properties of theIgG2M3 and IgG1 mutants.

Cell Culture:

Human Burkitt's lymphoma cell line Raji (American Type CultureCollection) was maintained in RPMI 1640 with L-glutamine (BioWhittaker™)containing 10% FBS (HyClone®) and 1% penicillin-streptomycin (LifeTechnologies®).

Antigen Binding Assays:

The antigen binding activity of OST577 wild-type and mutant antibodieswas confirmed in a competitive binding ELISA. Immulon™ 2 plates (DYNEX®Technologies) were coated overnight at 4° C. with 1.0 μg/ml ofrecombinant Hepatitis B Surface Antigen (HBsAg) (AdvancedImmunoChemical, Inc., Long Beach, Calif.). The next day, the plates werewashed with EWB and blocked with 300 μl/well of SuperBlock® BlockingBuffer in TBS (Pierce Chemical Company) for 30 minutes at roomtemperature. The plates were washed with EWB, and premixed biotinylatedOST577-IgG2M3 antibody (0.25 μg/ml) and competitor OST577-IgG2M3antibody (twofold serial dilutions from 33 μg/ml to 0.033 μg/ml) orpremixed biotinylated OST577-IgG1 antibody (0.25 μg/ml) and competitorOST577-IgG1 antibody (twofold serial dilutions from 67 μg/ml to 0.067μg/ml) in 100 μl of EB were added to each well. The plates wereincubated for 1 hour at room temperature, then washed with EWB, and 100μl/well of streptavidin-HRP conjugate (Pierce Chemical Company) wasadded at 1 μg/ml in EB. After incubation for 30 minutes at roomtemperature, the plates were washed with EWB, followed by addition of100 μl/well of ABTS Peroxidase Substrate/Peroxidase Solution B(Kirkegaard & Perry Laboratories). The reaction was stopped with 100μl/well of 2% oxalic acid, and the absorbance at 415 nm was measuredusing a VERSAmax™ microtitre plate reader (Molecular Devices®Corporation).

The antigen binding activity of Hu1D10-IgG2M3 wild-type and mutantantibodies was confirmed in a FACS™ binding assay using Raji cells,which express an allele of the HLA-DR β chain that is recognized byHu1D10 (Kostelny et al. (2001), op. cit.). Approximately 2.5×10⁵cells/test were washed once in FBB, pH 7.4, and resuspended in 140 μl ofHu1D10-IgG2M3 antibody (threefold serial dilutions from 60 μg/ml to0.027 μg/ml) in FBB, pH 7.4. The cells were incubated with antibody for1 hour on ice, washed twice in FBB, pH 7.4, and resuspended in 25 μl ofgoat F(ab′)₂ anti-human kappa RPE-conjugated antibody (SouthernBiotechnology Associates, Inc.) diluted to 10 μg/ml in FBB, pH 7.4.After incubation for 30 minutes on ice in the dark, the cells werewashed twice in FBB, pH 7.4, and resuspended in 1% formaldehyde. Sampleswere analyzed for antibody binding to the HLA-DR β chain allele by FACS™using a FACSCalibur flow cytometer (BD® Biosciences).

Similarly, the antigen binding activity of Hu1D10-IgG1 wild-type andmutant antibodies was confirmed in a FACS™ binding assay using Rajicells. Approximately 2.0×10⁵ cells/test were washed once in FBB, pH 7.4,and resuspended in 100 μl of Hu1D10-IgG1 antibody (twofold serialdilutions from 25 μg/ml to 12.5 μg/ml, then threefold serial dilutionsfrom 12.5 μg/ml to 0.0020 μg/ml) in FBB, pH 7.4. A dilution series ofHuFd79-IgG1 antibody (Co et al., Proc. Natl. Acad. Sci. 88:2869-2873(1991)) was prepared as described above and used as a negative control.The cells were incubated with antibody for 1 hour on ice, washed twicein FBB, pH 7.4, and resuspended in 25 μl of goat F(ab′)₂ anti-human IgGFITC-conjugated antibody (Southern Biotechnology Associates, Inc.)diluted to 20 μg/ml in FBB, pH 7.4. After incubation for 30 minutes onice in the dark, the cells were washed twice in FBB, pH 7.4, andresuspended in 1% formaldehyde. Samples were analyzed for antibodybinding to the HLA-DR β chain allele by FACS™ using a FACSCalibur flowcytometer (BD® Biosciences).

ADCC Assay:

The antibody-dependent cell-mediated cytotoxicity (ADCC) activity ofHu1D10 wild-type and mutant antibodies was confirmed by measuringlactate dehydrogenase (LDH) release using human peripheral bloodmononuclear cells (PBMC) as effectors and Raji cells as targetsfollowing a published method (Shields et al., op. cit.). PBMC wereprepared from fresh whole blood using a Ficoll-Paque® Plus gradient(Amersham Biosciences™ Corporation) and resuspended at a density of8×10⁶ cells/ml in assay medium (RPMI 1640, 1% BSA). Raji cells werewashed three times in assay medium and resuspended at a density of0.4×10⁶ cells/ml in assay medium. Hu1D10 wild-type and mutant antibodieswere diluted to 4 μg/ml, 0.25 μg/ml, and 0.016 μg/ml in assay medium.Raji cells (50 μl/well) and Hu1D10 antibody (50 μl/well, i.e., 200ng/test, 12.5 ng/test, or 0.8 ng/test) were combined in the wells of aFalcon 96-well U-bottom assay plate (BD® Biosciences) and incubated for30 minutes at room temperature. PBMC (100 μl/well, i.e., 40:1effector/target ratio) were added to the opsonized cells and incubatedfor 4 hours at 37° C. in a CO₂ incubator. Antibody independentcell-mediated cytotoxicity (AICC) was measured by incubating effectorand target cells in the absence of antibody. Spontaneous release wasmeasured by incubating target cells (SR_(target)) or effector cells(SR_(effector)) in the absence of antibody. Maximum release (MR) wasmeasured by adding 2% Triton X-100 to target cells. The plates weregently centrifuged, and the supernatants (100 μl/well) were transferredto a Falcon 96-well flat-bottom plate. LDH activity was measured byincubating the supernatants with 100 μl/well of LDH reaction mixturefrom the Cytotoxicity Detection Kit (Roche Diagnostics Corporation) for30 minutes at room temperature. The reaction was stopped with 50 μl/wellof 1 N HCl, and the absorbance at 490 nm was measured using a VERSAmax™microtitre plate reader (Molecular Devices® Corporation). The percentcytotoxicity was calculated as (LDHrelease_(sample)−SR_(effector)−SR_(target))/(MR_(target)−SR_(target))×100.

Results:

The antigen binding properties of wild-type and mutant OST577 and Hu1D10antibodies to their respective antigens were confirmed using appropriatebinding assays. As described above, the binding of OST577 antibodies toHBsAg was determined in a competitive ELISA. As shown in FIG. 16A, thebinding of the wild-type and mutant OST577-IgG2M3 antibodies to HBsAgwas essentially identical. Similarly, as shown in FIG. 16B, the bindingof the wild-type and mutant OST577-IgG1 antibodies to HBsAg wasessentially identical.

As described above, the binding of Hu1D10 antibodies to an allele of theHLA-DR β chain was determined in a FACS binding assay. As shown in FIG.17A, the binding of the wild-type and mutant Hu1D10-IgG2M3 antibodies tothe HLA-DR β chain allele was essentially identical. Similarly, as shownin FIG. 17B, the binding of the wild-type and mutant Hu1D10-IgG1antibodies to the HLA-DR β chain allele was essentially identical. Theseresults indicate that, as expected, the mutations described at positions250 and 428 do not affect antigen binding.

The ADCC activity of Hu1D10 wild-type and mutant antibodies wasconfirmed in an LDH release assay using human PBMC as effectors and Rajicells as targets. As shown in FIG. 18A, using a donor carryinghomozygous 158 V/V FcγRIII alleles, the ADCC activity of the doublemutant (T250Q/M428L) Hu1D10-IgG1 antibody was very similar to that ofthe wild-type antibody while the ADCC activity of the single mutant(M428L) Hu1D10-IgG1 antibody was slightly diminished compared to thewild-type antibody. As expected, the wild-type and mutant Hu1D10-IgG2M3antibodies lacked ADCC activity. Similarly, as shown in FIG. 18B, usinga donor carrying homozygous 158 F/F FcγRIII alleles, the ADCC activityof the double mutant (T250Q/M428L) Hu1D10-IgG1 antibody was very similarto that of the wild-type antibody while the ADCC activity of the singlemutant (M428L) Hu1D10-IgG1 antibody was somewhat diminished compared tothe wild-type antibody. The wild-type and mutant Hu1D10-IgG2M3antibodies lacked ADCC activity. These results indicate that theT250Q/M428L mutation described in this invention does not affect theADCC activity of the IgG1 form of the antibody, while the M428L mutationslightly reduces the ADCC activity of the IgG1 form. The mutationsdescribed at positions 250 and 428 do not affect the ADCC activity ofthe IgG2M3 form of the antibody.

Example 9

This example describes in vitro and in vivo serum half-life assays.

Human IgG antibodies with higher (or lower) affinity to FcRn in vitroare expected to have longer (or shorter) serum half-life in vivo,respectively. The affinity of human IgG mutants to FcRn may be measuredin vitro by various methods such as surface plasmon resonance (SPR)using soluble FcRn conjugated to a suitable biosensor chip, or byperforming a competitive binding experiment using FcRn expressed on thesurface of transfected cells. The FcRn used in the in vitro affinityexperiments may be of murine, rhesus, or human origin. The serumhalf-life of human IgG mutants with the desired properties may bemeasured in vivo by injecting suitable experimental animals (e.g., miceor monkeys) or humans with a dose of antibody in the range 0.1-10 mg ofantibody per kg of body weight, then withdrawing serum samples atvarious time intervals spanning the expected serum half-life of theantibody, and assaying the samples for the presence of intact IgG by asuitable technique such as ELISA.

Rhesus Pharmacokinetics Study:

A non-GLP pharmacokinetics study entitled “Pharmacokinetic Comparison ofThree Variants of OST577” was conducted at the California NationalPrimate Research Center (CNPRC) at the University of California, Davis.Twelve male rhesus macaques were randomized by weight and assigned toone of three study groups. The four animals comprising each study groupeach received a single intravenous dose of wild-type or one of twovariants of OST577 at 1 mg/kg administered over fifteen minutes. TheOST577 antibodies were wild-type OST577-IgG2M3, a variant ofOST577-IgG2M3 containing the single mutation M428L, and a variant ofOST577-IgG2M3 containing the double mutation T250Q/M428L. All threeantibodies were expressed by transfection of Sp2/0 cells and purified asdescribed in Example 5.

Blood samples were drawn prior to dosing on day 0, at 1 and 4 hoursafter dosing, and at 1, 7, 14, 21, 28, 42, and 56 days. At each timepoint, 4 mls of blood was drawn from a saphenous vein, serum wasprepared, and 2 aliquots were frozen and maintained at −20° C. untiluse. For serum chemistry and hematology determinations, blood sampleswere drawn 16 days prior to the study, prior to dosing on day 0, and atthe conclusion of the study on day 56.

ELISA:

The concentration of the OST577-IgG2M3 antibodies in rhesus serumsamples was determined by ELISA using a qualified assay. Pooled normalrhesus serum (PNRS) was obtained from the CNPRC. The same lot of PNRSwas used to prepare calibrators, positive serum controls, and forpre-dilution of rhesus serum samples. Calibrators were prepared bystandard dilution of OST577-IgG2M3 in PNRS at 3000, 1500, 750, 375,187.5, 93.75, 46.88, 23.44, and 0 ng/ml, equilibrated for 2 hours atroom temperature, and frozen in aliquots at −20° C. Positive serumcontrols were prepared by spiking PNRS with OST577-IgG2M3 at 0.2 μg/mlfor the low positive serum control, 0.4 μg/ml for the medium positiveserum control, and 0.8 μg/ml for the high positive serum control,equilibrated for 2 hours at room temperature, and frozen in aliquots at−20° C. Predose serum samples from each animal were used as negativeserum controls.

Immulon™ 2 plates (DYNEX® Technologies, Inc.) were coated overnight at2-8° C. with 100 μl/well of a mouse anti-OST577-IgG1 idiotype monoclonalantibody (OST577-γ1 anti-id, Protein Design Labs™, Inc.) at 1.0 μg/ml inPBS. The next day the plates were washed three times with 300 μl/well ofPBS/Tween (Phosphate Buffered Saline, 0.1% Tween 20), tapped dry on apaper towel, and blocked with 300 μl/well of SuperBlock® Blocking Bufferin PBS (Pierce Chemical Company) for 60±5 minutes at room temperature.Calibrators, positive and negative serum controls, and serum sampleswere thawed and brought to room temperature before use. Calibrators, andpositive and negative serum controls were diluted 1:10 in SuperBlock®Blocking Buffer in PBS. Serum samples were appropriately pre-diluted(1:10 to 1:80) in PNRS, then diluted 1:10 in SuperBlock® Blocking Bufferin PBS. The plates were washed three times with 300 μl/well of PBS/Tweenand tapped dry on a paper towel. Diluted calibrators, positive andnegative serum controls, and serum samples were then added at 100μl/well in duplicate wells and incubated for 60±5 minutes at roomtemperature. The plates were washed three times with 300 μl/well ofPBS/Tween and tapped dry on a paper towel. Goat anti-human lambda lightchain HRP-conjugated antibody (Southern Biotechnology Associates, Inc.)was prepared by 1:1000 dilution in PBS/BSA/Tween (Phosphate BufferedSaline, 0.5% Bovine Serum Albumin, 0.1% Tween 20), added at 100 μl/well,and incubated for 60±5 minutes at room temperature. The plates werewashed three times with 300 μl/well of PBS/Tween and tapped dry on apaper towel. ABTS Peroxidase Substrate/Peroxidase Solution B (Kirkegaard& Perry Laboratories) was added at 100 μl/well, and incubated for 7±1minutes. Development was stopped by addition of Substrate Stop Solution(2% Oxalic Acid) at 100 μl/well. Absorbance values at 415 nm weremeasured within 30 minutes after adding the Substrate Stop Solutionusing a VERSAmax™ microtitre plate reader (Molecular Devices®Corporation).

A calibration curve was prepared using the mean absorbance valuesobtained from the calibrators and fitting the data to a four parameterlogistic regression curve using SOFTmax® PRO, version 4.0 (MolecularDevices® Corporation). The mean absorbance value for the negative serumcontrol (i.e., predose sample mean for each animal) was subtracted fromeach absorbance value obtained for the calibrators. The positive serumcontrol concentrations were determined after subtracting the meanabsorbance value obtained for the negative serum control from eachabsorbance value obtained for the positive serum controls.Concentrations corresponding to the resulting mean absorbance valueswere derived by interpolation from the calibration curve. Theconcentrations of serum samples were determined by subtracting the meanabsorbance value of the negative serum control from the absorbance valueof each sample, averaging the resulting absorbance values, deriving theconcentration corresponding to the mean absorbance value byinterpolation from the calibration curve, and multiplying the resultingconcentration by the pre-dilution factor, if any, to arrive at the finalconcentration for each sample.

The estimated quantitative range of the assay was 0.10-0.90 μg/ml. Theassay was considered suitable when the following two conditions weremet: (1) the mean back-calculated concentration of all three calibratorsin the quantitative range was within 20% of their nominal value; and (2)the mean calculated results of four of six positive serum controls waswithin 30% of their nominal value, and at least one mean result fromeach concentration level was within 30% of its nominal value. Data fromplates that did not meet the above criteria were rejected. Data fromindividual serum samples was rejected when any of the following threeconditions was met: (1) the absorbance values in duplicate wellsdiffered from each other by more than 40%; (2) the mean calculatedconcentration was below the lower limit of quantitation (LLOQ) of theassay (0.10 μg/ml); (3) the mean calculated concentration was above theupper limit of quantitation (ULOQ) of the assay (0.90 μg/ml).

Results:

The serum antibody concentration data were fitted with a two-compartmentmodel using WinNonlin® Enterprise Edition, version 3.2 (Pharsight®Corporation, Mountain View, Calif.). The model assumes a first orderdistribution and first order elimination rate and fits the data well.The modeled data (simulated based on each group's geometric mean of theprimary pharmacokinetic parameters) as well as the observed mean serumantibody concentration (μg/ml) and the standard deviation for each groupof four animals were plotted as a function of time (days after infusion)using GraphPad Prism®, version 3.02 (GraphPad™ Software, Inc.). As shownin FIG. 19, the data indicate that the mean serum antibodyconcentrations of the M428L and T250Q/M428L variants of OST577-IgG2M3were maintained at higher levels than wild-type OST577-IgG2M3 at alltime points.

Various pharmacokinetic parameters were calculated from the data usingWinNonlin® Enterprise Edition, version 3.2 (Pharsight® Corporation).Statistical analyses of the pharmacokinetic parameters were calculatedusing GraphPad Prism®, version 3.02 (GraphPad™ Software, Inc.). As shownin Table 9, the mean maximum serum antibody concentration (Cmax) wasvery similar among the three test groups, indicating that theadministered antibodies were distributed to the circulation in a similarmanner. Thus, the higher antibody concentrations of the mutant IgG2M3antibodies following the distribution phase are attributable to theirincreased persistence in the serum. Analysis of the mean clearance (CL)indicated that this was the case. The mean CL, the volume of serumantibody cleared per unit of time, was approximately 1.8-fold lower forthe M428L variant (0.0811±0.0384 ml/hr/kg; p=0.057), and approximately2.8-fold lower for the T250Q/M428L variant (0.0514±0.0075 ml/hr/kg;p=0.029) compared to wild-type OST577-IgG2M3 (0.144±0.047 ml/hr/kg)(Table 9), indicating a significant decrease in the clearance of theOST577-IgG2M3 M428L and T250Q/M428L variants from the circulation ofrhesus monkeys compared to the wild-type.

The PK profiles of the OST577-IgG2M3 variants were further analyzed bycalculating other parameters (Table 9). Since the AUC (Area Under theCurve) is inversely proportional to CL, it follows that the mean AUC,the area under the concentration-time curve extrapolated from time zeroto infinity, was approximately 2-fold higher for the M428L variant(15,200±8,700 hr*μg/ml; p=0.057), and approximately 2.6-fold higher forthe T250Q/M428L variant (19,800±2,900 hr*μg/ml; p=0.029) compared towild-type OST577-IgG2M3 (7,710±3,110 hr*μg/ml) (Table 9), indicating asignificant increase in the total exposure of the OST577-IgG2M3 M428Land T250Q/M428L variants compared to the wild-type.

Finally, the mean elimination (β-phase) half-life was approximately1.8-fold longer for the M428L variant (642±205 hr), and approximately1.9-fold longer for the T250Q/M428L variant (652±28 hr; p=0.029)compared to wild-type OST577-IgG2M3 (351±121 hr) (Table 9). Theelimination half-life for wild-type OST577-IgG2M3 in this study issimilar to that for OST577-IgG1 (324±85 hr) in a previous PK study inrhesus monkeys (Ehrlich et al., op. cit.).

TABLE 9 CL^(c) AUC^(d) Elimination Name^(a) (IgG2M3) Cmax^(b) (μg/ml)(ml/hr/kg) (hr * μg/ml) half-life^(e) (hr) Wild-type 36.7 ± 12.8  0.144± 0.047  7710 ± 3110 351 ± 121 M428L 36.5 ± 20.1 0.0811* ± 0.0384 15200*± 8700 642 ± 205 T250Q/M428L 39.9 ± 6.8  0.0514* ± 0.0075 19800* ± 2900652* ± 28  ^(a)For each mutant, the first letter indicates the wild-typeamino acid, the number indicates the position according to the EU index(Kabat et al., op. cit.), and the second letter indicates the mutantamino acid. ^(b)Cmax values (±S.D.) are expressed in μg/ml and werecalculated from the PK data using WinNonlin as described in Example 9.^(c)CL values (±S.D.) are expressed in ml/hr/kg and were calculated fromthe PK data using WinNonlin as described in Example 9. ^(d)AUC values(±S.D.) are expressed in hr * μg/ml and were calculated from the PK datausing WinNonlin as described in Example 9. ^(e)Elimination half-lifevalues (±S.D.) are expressed in hr and were calculated from the PK datausing WinNonlin as described in Example 9. *Indicates a significantdifference (p < 0.060) between the wild-type group and each mutantgroup. Mann-Whitney tests were done using GraphPad Prism as described inExample 9.

Example 10

This example describes application of the in vitro and in vivo serumhalf-life assays described in Example 9 to mutants of IgG1 antibodies.

Rhesus Pharmacokinetics Study:

A non-GLP pharmacokinetics study entitled “Pharmacokinetic Comparison ofTwo Variants of OST577” was conducted at the California National PrimateResearch Center (CNPRC) at the University of California, Davis. Eightmale rhesus macaques were randomized by weight and assigned to one oftwo study groups. The four animals comprising each study group eachreceived a single intravenous dose of wild-type or a variant of OST577at 1 mg/kg administered over fifteen minutes. The OST577 antibodies werewild-type OST577-IgG1, and a variant of OST577-IgG1 containing thedouble mutation T250Q/M428L. Both antibodies were expressed bytransfection of Sp2/0 cells and purified as described in Example 5.

Blood samples were drawn prior to dosing on day 0, at 1 and 4 hoursafter dosing, and at 1, 7, 14, 21, 28, 42, and 56 days. At each timepoint, 4 mls of blood was drawn from a saphenous vein, serum wasprepared, and 2 aliquots were frozen and maintained at −20° C. untiluse. For serum chemistry and hematology determinations, blood sampleswere drawn prior to dosing on day 0, and at the conclusion of the studyon day 56.

ELISA:

The concentration of the OST577-IgG1 antibodies in rhesus serum sampleswas determined by ELISA using a validated assay. Pooled normal rhesusserum (PNRS) was obtained from the CNPRC. The same lot of PNRS was usedto prepare calibrators, positive and negative serum controls, and forpre-dilution of rhesus serum samples. Calibrators were prepared bystandard dilution of OST577-IgG1 in PNRS at 3200, 1600, 800, 400, 200,100, 50, 25, and 0 ng/ml, equilibrated for 2 hours at room temperature,and frozen in aliquots at −80° C. Positive serum controls were preparedby spiking PNRS with OST577-IgG1 at 0.2 μg/ml for the low positive serumcontrol, 0.4 μg/ml for the medium positive serum control, and 0.8 μg/mlfor the high positive serum control, equilibrated for 2 hours at roomtemperature, and frozen in aliquots at −80° C. PNRS was used as anegative serum control.

Immulon™ 2 plates (DYNEX® Technologies, Inc.) were coated overnight at2-8° C. with 100 μl/well of a mouse anti-OST577-IgG1 idiotype monoclonalantibody (OST577-γ1 anti-id, Protein Design Labs™, Inc.) at 1.0 μg/ml inPBS. The next day the plates were washed three times with 300 μl/well ofPBS/Tween (Phosphate Buffered Saline, 0.1% Tween 20), tapped dry on apaper towel, and blocked with 300 μl/well of SuperBlock® Blocking Bufferin PBS (Pierce Chemical Company) for 60±5 minutes at room temperature.Calibrators, positive and negative serum controls, and serum sampleswere thawed and brought to room temperature before use. Calibrators, andpositive and negative serum controls were diluted 1:10 in SuperBlock®Blocking Buffer in PBS. Serum samples were appropriately pre-diluted(1:5 to 1:80) in PNRS, then diluted 1:10 in SuperBlock® Blocking Bufferin PBS. The plates were washed three times with 300 μl/well of PBS/Tweenand tapped dry on a paper towel. Diluted calibrators, positive andnegative serum controls, and serum samples were then added at 100μl/well in duplicate wells and incubated for 60±5 minutes at roomtemperature. The plates were washed three times with 300 μl/well ofPBS/Tween and tapped dry on a paper towel. Goat anti-human lambda lightchain HRP-conjugated antibody (Southern Biotechnology Associates, Inc.)was prepared by 1:1000 dilution in PBS/BSA/Tween (Phosphate BufferedSaline, 0.5% Bovine Serum Albumin, 0.1% Tween 20), added at 100 μl/well,and incubated for 60±5 minutes at room temperature. The plates werewashed three times with 300 μl/well of PBS/Tween and tapped dry on apaper towel. ABTS Peroxidase Substrate/Peroxidase Solution B (Kirkegaard& Perry Laboratories) was added at 100 μl/well, and incubated for 7±1minutes. Development was stopped by addition of Substrate Stop Solution(2% Oxalic Acid) at 100 μl/well. Absorbance values at 415 nm weremeasured within 30 minutes after adding the Substrate Stop Solutionusing a VERSAmax™ microtitre plate reader (Molecular Devices®Corporation).

A calibration curve was prepared using the mean absorbance valuesobtained from the calibrators and fitting the data to a four parameterlogistic regression curve using SOFTmax® PRO, version 4.0 (MolecularDevices® Corporation). The mean absorbance value for the 0.0 ng/mLcalibrator was subtracted from each absorbance value obtained for theremaining calibrators. The positive serum control concentrations weredetermined after subtracting the mean absorbance value obtained for thenegative serum control from each absorbance value obtained for thepositive serum controls. Concentrations corresponding to the resultingmean absorbance values were derived by interpolation from thecalibration curve. The concentrations of serum samples were determinedby subtracting the mean absorbance value of the appropriate predosesample from the absorbance value of each study sample, averaging theresulting absorbance values, deriving the concentration corresponding tothe mean absorbance value by interpolation from the calibration curve,and multiplying the resulting concentration by the pre-dilution factor,if any, to arrive at the final concentration for each sample.

The estimated quantitative range of the assay was 0.10-0.90 μg/ml. Theassay was considered suitable when the following two conditions weremet: (1) the mean back-calculated concentration of all four calibratorsin the quantitative range was within 20% of their nominal value; and (2)the mean calculated results of four of six positive serum controls waswithin 30% of their nominal value, and at least one mean result fromeach concentration level was within 30% of its nominal value. Data fromplates that did not meet the above criteria were rejected. Data fromindividual serum samples was rejected when either of the followingconditions was met: (1) the mean calculated concentration was below thelower limit of quantitation (LLOQ) of the assay (0.10 μg/ml); or (2) themean calculated concentration was above the upper limit of quantitation(ULOQ) of the assay (0.90 μg/ml). If the calculated result of duplicatesamples differed by more than 40% from each other, the sample wasretested in a second independent assay. If the mean calculated result ofthe second assay was within 15% of the mean calculated result of thefirst assay for the sample in question, the mean calculated result ofthe first assay was used. Otherwise, the sample was retested in a thirdindependent assay, and the results of all three assays were averaged,unless one value could be removed by outlier testing. In this case, themean of the two remaining values was reported.

Results:

The serum antibody concentration data were fitted with a two-compartmentmodel using WinNonlin® Enterprise Edition, version 3.2 (Pharsight®Corporation, Mountain View, Calif.). The model assumes a first orderdistribution and first order elimination rate and fits the data well.The modeled data (simulated based on the median values of each group'sprimary pharmacokinetic parameters) as well as the observed mean serumantibody concentration (μg/ml) and the standard deviation for each groupof four animals were plotted as a function of time (days after infusion)using GraphPad Prism®, version 3.02 (GraphPad™ Software, Inc.). As shownin FIG. 20, the data indicate that the mean serum antibodyconcentrations of the T250Q/M428L variant of OST577-IgG1 were maintainedat higher levels than wild-type OST577-IgG1 at all time points.

Various pharmacokinetic parameters were calculated from the data usingWinNonlin® Enterprise Edition, version 3.2 (Pharsight® Corporation).Statistical analyses of the pharmacokinetic parameters were calculatedusing GraphPad Prism®, version 3.02 (GraphPad™ Software, Inc.). As shownin Table 10, the Cmax was very similar between both test groups,indicating that the administered antibodies were distributed to thecirculation in a similar manner. Thus, the higher antibodyconcentrations of the mutant IgG1 antibody following the distributionphase are attributable to its increased persistence in the serum.Analysis of the mean CL indicated that this was the case. The mean CLwas approximately 2.3-fold lower for the T250Q/M428L variant(0.0811±0.0191 ml/hr/kg; p=0.029) compared to wild-type OST577-IgG1(0.190±0.022 ml/hr/kg) (Table 10), indicating a significant decrease inthe clearance of the OST577-IgG1 T250Q/M428L variant from thecirculation of rhesus monkeys compared to the wild-type.

The PK profile of the OST577-IgG1 variant was further analyzed bycalculating other parameters (Table 10). The mean AUC was approximately2.4-fold higher for the T250Q/M428L variant (12,900±3,000 hr*μg/ml;p=0.029) compared to wild-type OST577-IgG1 (5,320±590 hr*μg/ml) (Table10), indicating a significant increase in the total exposure of theOST577-IgG1 T250Q/M428L variant compared to the wild-type.

Finally, the mean elimination (β-phase) half-life was approximately2.5-fold longer for the T250Q/M428L variant (838±187 hr; p=0.029)compared to wild-type OST577-IgG1 (336±34 hr) (Table 10). Theelimination half-life for wild-type OST577-IgG1 in this study is similarto that for OST577-IgG1 (324±85 hr) in a previous PK study in rhesusmonkeys (Ehrlich et al., op. cit.).

TABLE 10 Cmax^(b) CL^(c) AUC^(d) Elimination half-life^(e) Name^(a)(IgG1) (μg/ml) (ml/hr/kg) (hr * μg/ml) (hr) Wild-type 26.0 ± 4.1  0.190± 0.022 5320 ± 590 336 ± 34 T250Q/M428L 29.1 ± 3.7 0.0811* ± 0.019112900* ± 3000  838* ± 187 ^(a)For each mutant, the first letterindicates the wild-type amino acid, the number indicates the positionaccording to the EU index (Kabat et al., op. cit.), and the secondletter indicates the mutant amino acid. ^(b)Cmax values (±S.D.) areexpressed in μg/ml and were calculated from the PK data using WinNonlinas described in Example 10. ^(c)CL values (±S.D.) are expressed inml/hr/kg and were calculated from the PK data using WinNonlin asdescribed in Example 10. ^(d)AUC values (±S.D.) are expressed in hr *μg/ml and were calculated from the PK data using WinNonlin as describedin Example 10. ^(e)Elimination half-life values (±S.D.) are expressed inhr and were calculated from the PK data using WinNonlin as described inExample 10. *Indicates a significant difference (p < 0.060) between thewild-type group and each mutant group. Mann-Whitney tests were doneusing GraphPad Prism as described in Example 10.

Example 11

This example describes application of the various binding analysesdescribed in Examples 6 and 7 to mutants of IgG3 and IgG4 antibodies.

Mutagenesis:

The overlap-extension PCR method (Higuchi, op. cit.) was used togenerate site-directed amino acid substitutions at position 428 of theHu1D10-IgG3 heavy chain, or positions 250 and 428 of the Hu1D10-IgG4heavy chain (numbered according to the EU index of Kabat et al., op.cit.). An M428L mutant was generated in the Hu1D10-IgG3 heavy chain.Both an M428L and a T250QM428L mutant were generated in the Hu1D10-IgG4heavy chain.

Transfections:

The wild-type or mutant Hu1D10-IgG3 or Hu1D10-IgG4 heavy chainexpression vectors, described in detail in Examples 1 and 2, weretransiently co-transfected with the pVk-Hu1D10 light chain expressionvector into human kidney cell line 293-H (Life Technologies®). TheHu1D10-IgG3 or Hu1D10-IgG4 expression vectors were also stablyco-transfected with the pVk-Hu1D10 expression vector into Sp2/0 cells,as described in Example 5. For stable transfections in Sp2/0, the IgG3expression vectors were linearized with FspI; however, the IgG4expression vectors were linearized with BstZ171 since there are two FspIsites in pHuHC.g4.Tt.D-Hu1D10.

Antibody Purification:

Culture supernatants containing human IgG4 antibodies were quantified byELISA, harvested by centrifugation, sterile filtered, and purified byprotein A affinity chromatography, as described in Example 5.

Culture supernatants containing human IgG3 antibodies were quantified byELISA, harvested by centrifugation, and sterile filtered, as describedin Example 5. The pH of the filtered supernatants was adjusted byaddition of 1/75 volume of 1 M Tris-HCl, pH 8.0. Supernatants were runover a 1 ml HiTrap® Protein G HP column (Amersham Biosciences™Corporation) that was pre-equilibrated with 20 mM sodium phosphate, pH7.0. The column was washed with the same buffer, and bound antibody waseluted with 100 mM glycine-HCl, pH 2.7. After neutralization by additionof ˜ 1/50 volume of 1 M Tris-HCl, pH 8.0, the pooled protein fractionswere either dialyzed overnight in 20 mM sodium citrate, 120 mM NaCl, pH6.0, or run over a 5 ml HiTrap® Desalting column (Amersham Biosciences™Corporation) that was pre-equilibrated with 20 mM sodium citrate, 120 mMNaCl, pH 6.0. The flow-through from the desalting column was collectedand fractions with OD₂₈₀>0.1 were pooled and concentrated to ˜0.5-1.0mg/ml using 2 ml Vivaspin® concentrators (50,000 dalton MWCO)(Vivascience® AG). Dialyzed material was concentrated in the samemanner. Samples were then filter sterilized using 0.2 μm Millex®-GVmicrofilters (Millipore® Corporation). The concentrations of thepurified antibodies were determined by UV spectroscopy by measuring theabsorbance at 280 nm (1 mg/ml=1.4 A₂₈₀).

SDS-PAGE:

Five μg samples of purified antibodies were run under reducing ornon-reducing conditions, as described in Example 5.

Competitive Binding Assays:

As described in Example 6, NS0 transfectants expressing recombinant,GPI-linked human or rhesus FcRn on the surface were maintained inmycophenolic acid (MPA) selection medium (DMEM, 10% FBS, 1×HT MediaSupplement Hybri-Max® (Sigma®), 250 μg/ml xanthine (Sigma®), 1 μg/mlmycophenolic acid (Life Technologies®), and 2 mM L-glutamine) or 2×MPAselection medium.

A dilution series of each purified Hu1D10-IgG3 antibody was competedagainst human IgG (Sigma-Aldrich, St. Louis, Mo.) that had been labeledwith biotin (Pierce Biotechnology, Inc., Rockford, Ill.). Antibodieswere tested for binding to both human FcRn on cell line NS0 HuFcRn(memb), clone 7-3, and to rhesus FcRn on cell line NS0 RhFcRn, cloneR-3. Approximately 2×10⁵ cells/test were washed once in FBB, pH 8.0, andonce in FBB, pH 6.0, then resuspended in 120 μl of pre-mixedbiotinylated human IgG antibody (8.3 μg/ml) and Hu1D10-IgG3 competitorantibody (twofold serial dilutions from 625 μg/ml to 0.305 μg/ml) inFBB, pH 6.0. The cells were incubated with the antibody mixture for 1hour on ice, washed twice in FBB, pH 6.0, and resuspended in 25 μl ofstreptavidin-RPE conjugate (BioSource International) diluted to 2.5μg/ml in FBB, pH 6.0. After incubation for 30 minutes on ice in thedark, the cells were washed twice in FBB, pH 6.0, and resuspended in 1%formaldehyde. Samples were analyzed for antibody binding to FcRn byFACS™ using a FACSCalibur flow cytometer (BD® Biosciences).

A dilution series of each purified Hu1D10-IgG4 antibody was competedagainst human IgG (Sigma-Aldrich) that had been labeled with biotin(Pierce Biotechnology, Inc.). The IgG4 antibodies were tested forbinding to both human FcRn on cell line NS0 HuFcRn (memb), clone 7-3,and to rhesus FcRn on cell line NS0 RhFcRn, clone R-3, as describedabove for the IgG3 antibodies.

pH-Dependent Binding and Release Assay:

Purified Hu1D10-IgG3 and Hu1D10-IgG4 mutant antibodies were compared tothe respective wild-type antibodies for binding to human or rhesus FcRnand then released at various pH values in single-point binding andrelease assays using cell lines NS0 HuFcRn (memb), clone 7-3, and NS0RhFcRn, clone R-3, respectively, as described in Example 7. To ensurethat both subtypes, IgG3 and IgG4, were labeled equivalently, 25 μl ofgoat F(ab′)₂ anti-human kappa FITC-conjugated antibody (SouthernBiotechnology Associates, Inc.) diluted to 1.25 μg/ml in FBB of theappropriate pH was used as the detecting reagent.

Results:

Amino acid substitutions were generated at position 428 of the human γ3heavy chain and positions 250 and 428 of the human γ4 heavy chain(numbered according to the EU index of Kabat et al., op. cit.). Thesetwo positions were chosen based on the identification of mutations atthese positions in the human γ2M3 heavy chain that resulted in increasedbinding to FcRn. The M428L mutant (SEQ ID NO:115) was evaluated in thehuman γ3 heavy chain. Both the M428L mutant (SEQ ID NO:116) and theT250Q/M428L mutant (SEQ ID NO:117) were evaluated in the human γ4 heavychain.

The IgG3 and IgG4 Fc mutants were expressed as anti-HLA-DR β chainallele antibodies, comprising the light and heavy chain variable regionsof Hu1D10 (Kostelny et al. (2001), op. cit.), the light chain constantregion of human kappa (Hieter et al. (1980), op. cit.), and the heavychain constant regions of human IgG3 (Huck et al., op. cit.) and IgG4(Ellison et al, op. cit.), respectively. As described above, theappropriate wild-type or mutant heavy chain expression vector wastransiently co-transfected with the appropriate light chain expressionvector into 293-H cells for expression of Hu1D10 monoclonal antibodies.ELISA analysis of culture supernatants harvested 5-7 days aftertransient transfection showed that antibody expression levels weretypically 5-25 μg/ml in 25 ml of supernatant. The Hu1D10-IgG3 andHu1D10-IgG4 antibodies were purified by affinity chromatography usingprotein G and protein A, respectively, for a final yield ofapproximately 100-500 μg. Stable expression of Hu1D10 antibodies inSp2/0 cells typically resulted in expression levels of 30-100 μg/ml asdetermined by ELISA. Yields of approximately 50-80% of the antibodypresent in culture supernatants were obtained by small-scale protein Gor protein A affinity chromatography.

Purified antibodies were characterized by SDS polyacrylamide gelelectrophoresis (SDS-PAGE) under non-reducing and reducing conditions.SDS-PAGE analysis under non-reducing conditions indicated that thepurified antibodies had a molecular weight of about 150-170 kD (data notshown); analysis under reducing conditions indicated that the purifiedantibodies were comprised of a heavy chain with a molecular weight ofabout 50-60 kD and a light chain with a molecular weight of about 25 kD(data not shown). SDS-PAGE analysis of antibodies purified from stableSp2/0 transfectants gave results similar to those observed withantibodies purified from transient 293-H transfections.

The relative binding of wild-type Hu1D10-IgG3 and Hu1D10-IgG4 antibodiesand their various mutants to FcRn was determined using a transfected NS0cell line stably expressing human FcRn on its surface. As describedabove, purified antibodies were tested for FcRn binding in competitivebinding assays. Increasing concentrations of unlabeled competitorantibodies were incubated with cells in the presence of a sub-saturatingconcentration of biotinylated human IgG antibody (Sigma-Aldrich) in FBB,pH 6.0.

The binding of Hu1D10-IgG3 wild-type and the M428L mutant to human FcRnwere tested in competitive binding experiments. As summarized in Table11, the IC50 for the wild-type Hu1D10-IgG3 antibody is ˜15 μg/ml,whereas the IC50 for the M428L single mutant is ˜2 μg/ml. The binding ofHu1D10-IgG4 and its mutants to human FcRn were also tested incompetitive binding experiments. As summarized in Table 12, the IC50 forthe wild-type Hu1D10-IgG4 antibody is ˜76 μg/ml, whereas the IC50 forthe M428L single mutant is ˜5 μg/ml, and the IC50 for the T250Q/M428Ldouble mutant is ˜1 μg/ml.

The binding of Hu1D10-IgG3 wild-type and the M428L mutant to rhesus FcRnwere tested in competitive binding experiments. As summarized in Table13, the IC50 for the wild-type Hu1D10-IgG3 antibody is ˜14 μg/ml,whereas the IC50 for the M428L single mutant is ˜3 μg/ml. The binding ofHu1D10-IgG4 and its mutants to rhesus FcRn were also tested incompetitive binding experiments. As summarized in Table 14, the IC50 forthe wild-type Hu1D10-IgG4 antibody is ˜98 μg/ml, whereas the IC50 forthe M428L single mutant is ˜7 μg/ml, and the IC50 for the T250Q/M428Ldouble mutant is ˜1 μg/ml.

The binding of IgG to FcRn is known to be pH-dependent: IgG bindsstrongly to FcRn at pH 6.0 but weakly at pH 8.0. In order to engineermutant antibodies with longer serum half-lives, it is desirable toincrease binding to FcRn at pH 6.0, while retaining pH-dependent releasefrom FcRn at pH 8.0. To confirm that binding was pH-dependent, theantibodies were tested for binding to a transfected NS0 cell line stablyexpressing human FcRn and then released at pH values ranging from pH 6.0to pH 8.0. As described above, the cells were incubated with asub-saturating concentration of antibody in FBB, pH 6.0, washed withFBB, pH 6.0, 6.5, 7.0, 7.5, or 8.0, and binding was analyzed by FACS™.As shown in FIG. 21A, the results indicated that the modifiedHu1D10-IgG3 antibody with the M428L mutation showed strong binding tohuman FcRn at pH 6.0, with diminishing binding as the pH valuesincreased to pH 8.0. As shown in FIG. 21B, the results indicated thatthe modified Hu1D10-IgG4 antibodies with the M428L or T250Q/M428Lmutations both showed strong binding to human FcRn at pH 6.0, withdiminishing binding as the pH values increased to pH 8.0. These resultsindicated that the binding of the IgG3 and IgG4 antibodies to human FcRnwas pH-dependent.

Similarly, the antibodies were tested for binding to a transfected NS0cell line stably expressing rhesus FcRn and then released at pH valuesranging from pH 6.0 to pH 8.0. As shown in FIG. 21C, the resultsindicated that the modified Hu1D10-IgG3 antibody with the M428L mutationshowed strong binding to rhesus FcRn at pH 6.0, with diminishing bindingas the pH values increased to pH 8.0. As shown in FIG. 21D, the resultsindicated that the modified Hu1D10-IgG4 antibodies with the M428L orT250Q/M428L mutations showed strong binding to rhesus FcRn at pH 6.0,with diminishing binding as the pH values increased to pH 8.0. Theseresults indicated that the binding of the antibodies to rhesus FcRn wasalso pH-dependent.

TABLE 11 Name^(a) (IgG3) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 3 14.5 ± 3.6  1.0 M428L 3 2.29 ± 0.39 6.3 ^(a)For each mutant,the first letter indicates the wild-type amino acid, the numberindicates the position according to the EU index (Kabat et al., op.cit.), and the second letter indicates the mutant amino acid. ^(b)nindicates the number of independent assays. ^(c)IC50 values (±S.D.) areexpressed in μg/ml (based on final competitor concentrations) and werecalculated from competitive binding assays versus biotinylated human IgG(Sigma-Aldrich) in FBB, pH 6.0, as described in Example 11. ^(d)Relativebinding to human FcRn was calculated as the ratio of the IC50 value ofthe wild-type Hu1D10-IgG3 to that of the mutant.

TABLE 12 Name^(a) (IgG4) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 3 76.1 ± 12.7 1.0 M428L 3 5.03 ± 0.45 15 T250Q/M428L 3 1.07 ±0.14 71 ^(a)For each mutant, the first letter indicates the wild-typeamino acid, the number indicates the position according to the EU index(Kabat et al., op. cit.), and the second letter indicates the mutantamino acid. ^(b)n indicates the number of independent assays. ^(c)IC50values (±S.D.) are expressed in μg/ml (based on final competitorconcentrations) and were calculated from competitive binding assaysversus biotinylated human IgG (Sigma-Aldrich) in FBB, pH 6.0, asdescribed in Example 11. ^(d)Relative binding to human FcRn wascalculated as the ratio of the IC50 value of the wild-type Hu1D10-IgG4to that of each of the mutants.

TABLE 13 Name^(a) (IgG3) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 3 13.8 ± 1.0  1.0 M428L 3 3.03 ± 0.44 4.6 ^(a)For each mutant,the first letter indicates the wild-type amino acid, the numberindicates the position according to the EU index (Kabat et al., op.cit.), and the second letter indicates the mutant amino acid. ^(b)nindicates the number of independent assays. ^(c)IC50 values (±S.D.) areexpressed in μg/ml (based on final competitor concentrations) and werecalculated from competitive binding assays versus biotinylated human IgG(Sigma-Aldrich) in FBB, pH 6.0, as described in Example 11. ^(d)Relativebinding to rhesus FcRn was calculated as the ratio of the IC50 value ofthe wild-type Hu1D10-IgG3 to that of the mutant.

TABLE 14 Name^(a) (IgG4) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 3 98.4 ± 15.8 1.0 M428L 3 6.64 ± 1.05 15 T250Q/M428L 3 1.27 ±0.13 77 ^(a)For each mutant, the first letter indicates the wild-typeamino acid, the number indicates the position according to the EU index(Kabat et al., op. cit.), and the second letter indicates the mutantamino acid. ^(b)n indicates the number of independent assays. ^(c)IC50values (±S.D.) are expressed in μg/ml (based on final competitorconcentrations) and were calculated from competitive binding assaysversus biotinylated human IgG (Sigma-Aldrich) in FBB, pH 6.0, asdescribed in Example 11. ^(d)Relative binding to rhesus FcRn wascalculated as the ratio of the IC50 value of the wild-type Hu1D10-IgG4to that of each of the mutants.

Example 12

This example describes application of the in vitro and in vivo serumhalf-life assays described in Examples 9 and 10 to mutants of IgG3 andIgG4 antibodies.

The protocols of the “Rhesus Pharmacokinetics Study” as described inExamples 9 and 10 are carried out on mutants of IgG3 and IgG4 to confirmthe effect of the mutations on in vivo serum half-life and the variouspharmacokinetic parameters.

Example 13

This example describes the design and production of FcRn binding mutantsof well-known therapeutic antibodies thereby extending (or decreasing)the serum half-life of each so as to allow improved patient treatmentregimens.

Daclizumab:

Daclizumab is a humanized anti-CD25 monoclonal antibody that isundergoing clinical development for a number of autoimmune andinflammatory disease indications including asthma, diabetes, uveitis,multiple sclerosis, rheumatoid arthritis and ulcerative colitis.Daclizumab is currently marketed under the trademark Zenapax® fortransplant indications only.

The amino acid sequences of the light and heavy chain variable regionsfor daclizumab have been disclosed in U.S. Pat. No. 5,530,101, which ishereby incorporated by reference herein.

Daclizumab in its current clinical embodiment is an IgG1 isotypeantibody, however, an IgG2M3 isotype version of daclizumab may beproduced that exhibits similar therapeutic characteristics.

In order to increase the serum half-life of daclizumab, its constantregion amino acid sequence may be modified with any of the FcRn bindingmutations described above. For example, the T250Q/M428L mutation may beproduced in daclizumab using the methods for vector design andmutagenesis described in Examples 1-4 above. The sequence of T250Q/M428Ldaclizumab is depicted in FIG. 22 (SEQ ID NO: 122).

The increased serum half-life of T250Q/M428L daclizumab may bedetermined using the in vitro binding assay methods described inExamples 5-7, or using the in vivo assay methods described in Examples9-10.

It is expected that T250Q/M428L daclizumab will provide the therapeuticbenefits of the unaltered daclizumab with the advantage of decreasedfrequency and amount of dosage.

The other FcRn binding mutations in daclizumab (IgG1 isotype) constantregion sequences, as depicted in FIG. 22 (SEQ ID NOs:119-123), may alsobe produced according to the methods of Examples 1-4 above. It isexpected that these mutants will also affect serum half-life ofdaclizumab as desired based on the effects of these mutations in theOST577 and Hu1D10 antibody examples above.

Similarly, the FcRn binding mutation in an IgG2M3 version of daclizumabconstant region sequences, as depicted in FIG. 22 (SEQ ID NOs:124-128),may also be produced according to the methods of Examples 1-4 above.

Fontolizumab:

Fontolizumab is a humanized anti-interferon-gamma (IFN-γ) monoclonalantibody of the IgG1 isotype also known as HuZAF™. Fontolizumab iscurrently undergoing clinical development as a therapeutic treatment forCrohn's disease. The variable region sequences of fontolizumab aredisclosed in U.S. Pat. No. 6,329,511 which is hereby incorporated byreference herein. As described above for daclizumab, the serum half-lifeof fontolizumab may also be altered by mutating its constant regionsequences as shown in FIG. 23 (SEQ ID NOs:130-134), using the methodsdescribed above in Examples 1-4.

Visilizumab:

Visilizumab is a humanized anti-CD3 monoclonal antibody of the IgG2M3isotype, also known as Nuvion®. Visilizumab targets CD3 molecules, whichform the antigen-receptor complex on all mature T cells. Visilizumab iscurrently undergoing clinical development as treatment of steroidrefractory ulcerative colitis. As described above for daclizumab andfontolizumab, the serum half-life of visilizumab may also be altered bymutating its constant region sequences as shown in FIG. 24 (SEQ IDNOs:136-140), using the methods described above in Examples 1-4.

Volociximab:

Volociximab is a chimeric IgG4 antibody, also known as M200, directed tothe α5β1 integrin that is currently being developed as an angiogenesisinhibitor therapy directed to various proliferative disorders. Asdescribed above for daclizumab, fontolizumab, and visilizumab, the serumhalf-life of volociximab may also be altered by mutating its constantregion sequences as shown in FIG. 25 (SEQ ID NOs:142-146), using themethods described above in Examples 1-4.

Example 14

This example describes application of the various binding analysesdescribed in Examples 7 and 11 to mutants of the humanized anti-CD25antibody, daclizumab, in both IgG1 and IgG2M3 isotypes.

Mutagenesis:

Amino acid substitutions at positions 250 and 428 of the daclizumab(Dac) IgG1 and IgG2M3 heavy chains (numbered according to the EU indexof Kabat et al., op. cit.) were generated following the methodsdescribed in Examples 3-4 above. The T250Q/M428L mutation wasincorporated into Dac-IgG1 and Dac-IgG2M3 expression vectors using themethods for vector design described in Examples 1-2. Wild-type Dac-IgG1and Dac-IgG2M3 expression vectors were also constructed. The sequencesof the T250Q/M428L variants of daclizumab are depicted in FIG. 22 (SEQID NOS: 118, 122, and 127).

Transfections:

The Dac-IgG1 wild-type (WT), Dac-IgG2M3 WT, Dac-IgG1 T250Q/M428L, andDac-IgG2M3 T250Q/M428L expression vectors were stably transfected intoNS0 cells, as described in Example 6. The highest antibody-producingclones were chosen for expansion and adaptation to Protein-Free BasalMedium-2 (PFBM-2) (Protein Design Labs™, Inc.). The Dac-IgG1 WT,Dac-IgG1 T250Q/M428L, and Dac-IgG2M3 T250Q/M428L cell lines werecultured in 10 L bioreactors using a fed-batch process (Sauer et al.,op. cit.), supplemented volumetrically beginning 2 days afterinoculation with Protein-Free Feed Medium-3 (PFFM-3) (Protein DesignLabs™, Inc.), and harvested after 10-13 days. The Dac-IgG2M3 WT cellline was expanded to roller bottles in PFBM-2, supplemented after 2 dayswith 1/10 volume of PFFM-3, and grown to exhaustion. Other protein-freeor protein-containing media may also be suitable to support the cellcultures to provide the antibodies.

Antibody Purification:

Culture supernatants containing Dac-IgG1 WT, Dac-IgG1 T250Q/M428L, andDac-IgG2M3 T250Q/M428L antibodies were purified by protein A affinitychromatography, as follows. The cell culture harvest was adjusted to pH5.0 using 0.5 M citric acid, clarified by centrifugation, and adjustedto neutral pH. The clarified supernatants were purified by protein Aaffinity chromatography on a MabSelect Protein A column (GE™ Healthcare,Piscataway, N.J.) using methods similar to those described above inExample 5. The concentrations of the purified antibodies were determinedby UV spectroscopy by measuring the absorbance at 280 nm (1 mg/ml=1.34A₂₈₀).

SDS-PAGE:

Five μg samples of purified antibodies were run under reducing ornon-reducing conditions, as described in Example 5.

Competitive Binding Assays:

Antibodies were tested for binding to human FcRn on cell line NS0 HuFcRn(memb), clone 7-3, according to the methods described in Example 11.

pH-Dependent Binding and Release Assay:

The Dac-IgG1 T250Q/M428L and Dac-IgG2M3 T250Q/M428L mutant antibodieswere compared to the respective wild-type antibodies for binding tohuman FcRn and then released at various pH values in single-pointbinding and release assays using cell line NS0 HuFcRn (memb), clone 7-3,as described in Example 7.

Results:

Amino acid substitutions were generated at positions 250 and 428(numbered according to the EU index of Kabat et al., op. cit.) of thehuman γ1 and γ2M3 heavy chains of daclizumab. These two positions werechosen based on the identification of mutations at these positions inthe human γ1 and γ2M3 heavy chains of OST577 antibodies that resulted inincreased binding affinities to FcRn and longer serum half-lives.

Purified antibodies were characterized by SDS-PAGE under non-reducingand reducing conditions. SDS-PAGE analysis under non-reducing conditionsindicated that the purified antibodies had a molecular weight of about150-160 kD (data not shown); analysis under reducing conditionsindicated that the purified antibodies were comprised of a heavy chainwith a molecular weight of about 50 kD and a light chain with amolecular weight of about 25 kD (data not shown).

The relative binding of wild-type Dac-IgG1 and Dac-IgG2M3 antibodies andtheir respective T250Q/M428L mutants to FcRn was determined using atransfected NS0 cell line stably expressing human FcRn on its surface.The purified antibodies were tested for FcRn binding in competitivebinding assays. Increasing concentrations of unlabeled competitorantibodies were incubated with cells in the presence of a sub-saturatingconcentration of biotinylated human IgG antibody (Sigma-Aldrich) in FBB,pH 6.0. As summarized in Table 15, the IC50 for the wild-type Dac-IgG1antibody is ˜20 μg/ml, whereas the IC50 for the T250Q/M428L doublemutant is ˜0.6 μg/ml. As summarized in Table 16, the IC50 for thewild-type Dac-IgG2M3 antibody is ˜71 μg/ml, whereas the IC50 for theT250Q/M428L double mutant is ˜4 μg/ml.

To confirm that binding was pH-dependent, the antibodies were tested forbinding to a transfected NS0 cell line stably expressing human FcRn andthen released at pH values ranging from pH 6.0 to pH 8.0. The cells wereincubated with a sub-saturating concentration of antibody in FBB, pH6.0, washed with FBB, pH 6.0, 6.5, 7.0, 7.5, or 8.0, and binding wasanalyzed by FACS™. The results indicated that wild-type Dac-IgG1 andDac-IgG2M3 antibodies and the modified Dac-IgG1 and Dac-IgG2M3antibodies with the T250Q/M428L mutation all showed strong binding tohuman FcRn at pH 6.0, with diminishing binding as the pH valuesincreased to pH 8.0. These results confirmed that the binding of theDac-IgG1 and Dac-IgG2M3 antibodies to human FcRn was pH-dependent.

TABLE 15 Name^(a) (IgG1) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 4 20.4 ± 1.6  1.0 T250Q/M428L 3 0.626 ± 0.136 33 ^(a)For eachmutant, the first letter indicates the wild-type amino acid, the numberindicates the position according to the EU index (Kabat et al., op.cit.), and the second letter indicates the mutant amino acid. ^(b)nindicates the number of independent assays. ^(c)IC50 values (±S.D.) areexpressed in μg/ml (based on final competitor concentrations) and werecalculated from competitive binding assays versus biotinylated human IgG(Sigma-Aldrich) in FBB, pH 6.0, as described in Example 14. ^(d)Relativebinding to human FcRn was calculated as the ratio of the IC50 value ofthe wild-type Dac-IgG1 to that of the mutant.

TABLE 16 Name^(a) (IgG2M3) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 3 71.4 ± 5.0  1.0 T250Q/M428L 3 4.02 ± 0.56 18 ^(a)For eachmutant, the first letter indicates the wild-type amino acid, the numberindicates the position according to the EU index (Kabat et al., op.cit.), and the second letter indicates the mutant amino acid. ^(b)nindicates the number of independent assays. ^(c)IC50 values (±S.D.) areexpressed in μg/ml (based on final competitor concentrations) and werecalculated from competitive binding assays versus biotinylated human IgG(Sigma-Aldrich) in FBB, pH 6.0, as described in Example 14. ^(d)Relativebinding to human FcRn was calculated as the ratio of the IC50 value ofthe wild-type Dac-IgG2M3 to that of the mutant.

Example 15

This example describes application of the in vitro and in vivoelimination half-life assessment described in Examples 9 and 10 tomutants of the humanized anti-CD25 antibody, daclizumab, in both IgG1and IgG2M3 isotypes.

Cynomolgus Pharmacokinetics Study:

A non-GLP study of the pharmacokinetics of Dac-IgG1 WT, Dac-IgG1T250Q/M428L, and Dac-IgG2M3 T250Q/M428L following intravenousadministration to cynomolgus monkeys was conducted at Covance ResearchProducts, Inc., Alice, Tex. Thirty male cynomolgus macaques wereassigned, based on weight and the baseline level of CD25⁺ CD4⁺ T-cells,to one of three study groups. The ten animals comprising each studygroup each received a single intravenous slow bolus push of wild-type(Dac-IgG1 WT, group 1) or a variant of daclizumab (Dac-IgG1 T250Q/M428L,group 2; and Dac-IgG2M3 T250Q/M428L, group 3) at 10 mg/kg administeredwithin 2 minutes. All three antibodies were expressed by transfection ofNS0 cells and purified as described in Example 14.

Blood samples were drawn for determination of daclizumab serumconcentration levels prior to dosing on day 0 (predose), at 0.25, 1, 2,and 8 hours after dosing, and at 1, 2, 7, 10, 14, 18, 21, 28, 35, 42,49, and 56 days. At each time point, approximately 1 ml of blood wasdrawn via a femoral vein. Serum was prepared and serum samples werefrozen and maintained at approximately −70° C. until use. Blood samples(approximately 1 ml) were also drawn for the assessment of antibodies todaclizumab (anti-Ab). These samples were drawn at day 0 (predose), andat 14, 21, 28, 35, 42, 49, and 56 days. For serum chemistry andhematology determinations, blood samples were drawn prior to dosing onday 0, and at the conclusion of the study on day 56.

ELISA for Quantification of Serum Daclizumab:

The concentration of daclizumab (including the variant forms) in serumsamples was determined by ELISA. Calibrators and positive qualitycontrols (QC) were prepared by standard dilution of daclizumab in poolednormal cynomolgus serum (PNCS) (Bioreclamation, Inc., East Meadow, N.Y.)at 25, 50, 100, 250, 500, 1000, 2500, and 5000 ng/ml (calibrators); andat 50, 250, and 1000 ng/ml (QC). Separate calibrators and QC's wereprepared with each of the three daclizumab materials, equilibrated atroom temperature for 2 hours, and frozen in aliquots at −80° C. Duringsample testing, the calibrators and QC's used for each plate werematched to the serum samples of animals being tested on that plate. Forexample, samples from animals dosed with Dac-IgG1 WT (Group 1) weretested on plates including calibrators and controls prepared withDac-IgG1 WT.

Nunc Maxisorp™ 96-well microtiter plates (Nalge Nunc International,Rochester, N.Y.) were coated overnight at 2-8° C. with 100 μl/well ofIL-2 Receptor (R&D Systems™, Minneapolis, Minn.) at 25 ng/ml in sodiumcarbonate buffer. The next day the plates were emptied and tapped dry ona paper towel, and blocked with 300 μl/well of Super Block (ScyTekLaboratories, Inc., Logan, Utah) for 60±5 minutes at room temperature.Calibrators, positive and negative serum controls, and serum sampleswere thawed and brought to room temperature before use, then diluted 1:5in Super Block. Serum samples were first appropriately pre-diluted intothe assay quantitative range (1:2 to 1:1000) in PNCS, then diluted 1:5in Super Block. The plates were washed two times with 300 μl/well ofPBS/Tween (PBS, 0.05% Tween 20) and tapped dry on a paper towel. Dilutedcalibrators, positive and negative serum controls, and serum sampleswere then added at 100 μl/well in duplicate wells and incubated for 60±5minutes at room temperature. The plates were washed two times with 300μl/well of PBS/Tween and tapped dry on a paper towel. Sheep anti-humanIgG1 HRP-conjugated antibody (The Binding Site™, Inc., San Diego,Calif.) was diluted 1:3500 in PBS/BSA/Tween (Phosphate Buffered Saline,1% Bovine Serum Albumin, 0.05% Tween 20) as the detection reagent forsamples from animals dosed with Dac-IgG1 WT and Dac-IgG1 T250Q/M428L.Goat anti-human IgG (H+ L) antibody (Zymed™ Laboratories, Inc., SouthSan Francisco, Calif.) was diluted 1:5000 in PBS/BSA/Tween as thedetection reagent for samples from animals dosed with Dac-IgG2M3T250Q/M428L. The detection reagents were added at 150 μl/well, andincubated for 60±5 minutes at room temperature. The plates were washedsix times with 300 μl/well of PBS/Tween and tapped dry on a paper towel.TMB substrate (Sigma®) was added at 150 μl/well, and incubated for 10±1minute. Development was stopped by addition of Substrate Stop Solution(2.5 M Sulfuric Acid) at 50 μl/well. Absorbance values at 450 nm weremeasured within 30 minutes after adding the Substrate Stop Solutionusing a Spectramax™ microtiter plate reader (Molecular Devices®Corporation).

A calibration curve was prepared using the mean absorbance valuesobtained from the calibrators and fitting the data to a four parameterlogistic regression curve using SOFTmax® PRO, version 4.0 (MolecularDevices® Corporation). The mean absorbance value for the 0.0 ng/mlcalibrator was subtracted from each absorbance value obtained for theremaining calibrators. The positive serum QC concentrations weredetermined after subtracting the mean absorbance value obtained for thenegative QC from each absorbance value obtained for the positive serumcontrols. Concentrations corresponding to the resulting mean absorbancevalues were derived by interpolation from the calibration curve. Theconcentrations of serum samples were determined by subtracting the meanabsorbance value of the appropriate predose sample from the absorbancevalue of each study sample, averaging the resulting absorbance values,deriving the concentration corresponding to the mean absorbance value byinterpolation from the calibration curve, and multiplying the resultingconcentration by the pre-dilution factor, if any, to arrive at the finalconcentration for each sample.

The estimated quantitative range of the assay was established as 50ng/ml (LLOQ) to 1000 ng/ml (ULOQ). Each assay run was consideredacceptable when the following two conditions were met: (1) the meanback-calculated concentration of 4 of 5 calibrators in the quantitativerange (50, 100, 250, 500, and 1000 ng/ml) was within 20% of theirnominal value; and (2) the mean calculated results of four of sixpositive quality controls was within 30% of their nominal value, and atleast one mean result from each concentration level was within 30% ofits nominal value. Data from plates that did not meet the above criteriawere rejected. Data from individual serum samples was rejected wheneither of the following conditions was met: (1) the mean calculatedconcentration of a pre-diluted sample was below the LLOQ of the assay(50 ng/ml); or (2) the mean calculated concentration was above the ULOQof the assay (1000 ng/ml). If the calculated result of duplicate samplewells differed by more than 40%, the sample was retested.

ELISA Analysis for Anti-Daclizumab Antibodies:

ELISA methods were developed to detect and quantify anti-daclizumabantibodies when present in the serum of study animals. A murineanti-idiotypic antibody (anti-id) against daclizumab (Zid 5.1; ProteinDesign Labs™, Inc.) was used to prepare calibrators and positive QC's inPNCS (Bioreclamation, Inc.). Calibrators were prepared at 50, 100, 250,500, 1000, and 2500 ng/ml, and QC's were prepared at 300 and 600 ng/mlof anti-id. An un-spiked negative QC containing PNCS was also includedin each assay. Calibrators and controls were equilibrated for 2 hours atroom temperature, and frozen in aliquots at −80° C.

Nunc Maxisorp™ 96-well microtiter plates (Nalge Nunc International) werecoated overnight at 2-8° C. with 100 μl/well of daclizumab at 0.2 μg/mlin PBS. The next day the plates were emptied and tapped dry on a papertowel, and blocked with 300 μl/well of PBS/BSA (Phosphate BufferedSaline, 1% Bovine Serum Albumin) for 60±5 minutes at room temperature.Calibrators, positive and negative serum controls, and serum sampleswere thawed and brought to room temperature, then diluted 1:5 in SuperBlock (Scytek Laboratories). The plates were washed two times with 300μl/well of PBS/Tween and tapped dry on a paper towel. Dilutedcalibrators, positive and negative serum controls, and serum sampleswere then added at 100 μl/well in duplicate wells and incubated for 60±5minutes at room temperature. The plates were washed two times with 300μl/well of PBS/Tween and tapped dry on a paper towel. HRP-conjugateddaclizumab (Protein Design Labs™, Inc.) was diluted in PBS/BSA/Tween,added at 150 μl/well, and incubated for 60±5 minutes at roomtemperature. The plates were washed six times with 300 μl/well ofPBS/Tween and tapped dry on a paper towel. Enhanced K-Blue® SubstrateSolution (TMB) (Neogen™ Corporation, Lansing, Mich.) was added at 150μl/well, and incubated for 10-12 minutes. Development was stopped byaddition of Substrate Stop Solution (2.5 M Sulfuric Acid) at 50 μl/well.Absorbance values at 450 nm were measured within 30 minutes after addingthe Substrate Stop Solution using a Spectramax™ microtiter plate reader(Molecular Devices® Corporation).

During sample testing, the specific daclizumab coating antibody anddaclizumab-HRP detection reagent used for each plate was matched to theserum samples of animals being tested on that plate. For example,samples from animals dosed with Dac-IgG1 T250Q/M428L (Group 2) weretested on plates coated with Dac-IgG1 T250Q/M428L, and detected withDac-IgG1 T250Q/M428L-HRP. The dilutions used for the Dac-HRP conjugatesduring sample testing were 1:1000, 1:3000, or 1:6500 for Dac-IgG1WT-HRP, Dac-IgG1 T250Q/M428L-HRP, and Dac-IgG2M3 T250Q/M428L-HRP,respectively.

A calibration curve was prepared using the mean absorbance valuesobtained from the calibrators and fitting the data to a four parameterlogistic regression curve using SOFTmax® PRO, version 4.0 (MolecularDevices® Corporation). The mean absorbance value for the 0.0 ng/mlcalibrator was subtracted from each absorbance value obtained for theremaining calibrators. The positive QC concentrations were determinedafter subtracting the mean absorbance value obtained for the negative QCfrom each absorbance value obtained for the positive QC. Concentrationscorresponding to the resulting mean absorbance values were derived byinterpolation from the calibration curve. The concentrations of serumsamples were determined by subtracting the mean absorbance value of theappropriate predose sample from the absorbance value of each studysample, averaging the resulting absorbance values, deriving theconcentration corresponding to the mean absorbance value byinterpolation from the calibration curve, and multiplying the resultingconcentration by the pre-dilution factor, if any, to arrive at the finalconcentration for each sample.

The estimated quantitative range of the assay was determined to bebetween 100 ng/ml (LLOQ) and 2500 ng/ml (ULOQ) of anti-idiotypeequivalents. Sample result concentrations equal to or greater than theLLOQ, and equal to or less than the ULOQ were reported in relativeanti-idiotype equivalent concentration units. Samples withconcentrations >2500 ng/ml were reported as “>2500 ng/ml,” withoutsubsequent dilution and retesting. Results below the quantitative rangewere reported as “BQL” (below quantitative limits). Each assay run wasconsidered acceptable if the mean calculated concentration of the lowand high positive QC's was within 60% of their nominal concentration.Data from plates that did not meet this criterion were rejected, and allsamples from that run were retested. If absorbance values from duplicatesample wells differed by more than 25%, the sample was retested.

Results:

Among all 30 animals, 9, 6, and 8 animals from groups 1, 2, and 3,respectively, had detectable anti-daclizumab antibodies (anti-Ab).Although the timing of the detectable anti-Ab varied, the majority ofthe anti-Ab responses occurred after day 28 and were neutralizing innature as confirmed by the concurrent loss of daclizumab binding to CD25on peripheral CD4⁺ T-cells (data not shown). These anti-Ab responsescoincided with a significant reduction in serum daclizumabconcentrations indicating that these immunogenic responses significantlyaccelerated the drug clearance after they became detectable. Onestrategy to minimize the impact of the immunogenicity response on drugelimination is to truncate the data to a time before substantial anti-Abresponses are detected. This truncation cutoff time was determined to beday 28 based on two justifications: 1) the majority of the animals hadno detectable anti-Ab response until after the day 28 time point; 2) byday 28, the area under drug concentration-time curve (AUC_(0-28 day))was at least 80% of the total AUC (AUC extrapolated to infinity), hencethe degree of extrapolation in estimating the elimination half-life wasminimized and acceptable.

Several animals (n=3, 1, and 3 for groups 1, 2, and 3, respectively)developed high levels of anti-Ab on or earlier than day 28. The impactof the anti-Ab response on the PK profile is so profound in theseanimals that the evaluation of PK characteristics in these animalscannot be properly done even if the data were truncated to day 28 (datanot shown). Hence, these animals were excluded from the data analyses.As a result, 7, 9, and 7 animals were used in the data analyses andstatistics for groups 1, 2, and 3, respectively.

The serum antibody concentration data were fitted with a two-compartmentmodel using WinNonlin® Enterprise Edition, version 4.0.1 (Pharsight®Corporation). The model assumes a first order distribution and firstorder elimination rate and fits the data well. The modeled data(simulated based on the mean values of each group's primarypharmacokinetic parameters) as well as the observed mean serum antibodyconcentration (μg/ml) and the standard deviation for each group ofevaluable animals were plotted as a function of time (days afterinfusion) using GraphPad Prism®, version 3.02 (GraphPad™ Software,Inc.). As shown in FIG. 26, the concentration data indicated that themean serum antibody concentrations of both isotype mutant variants weremaintained at higher levels than the wild-type Dac-IgG1 antibody laterin the elimination phase.

Various pharmacokinetic parameters were calculated from data up to day28 using WinNonlin® Enterprise version, 4.0.1 (Pharsight® Corporation).Pair-wise comparisons of PK parameters between the Dac-IgG1 WT andDac-IgG1 T250Q/M428L mutant antibodies or between the Dac-IgG1 WT andDac-IgG2M3 T250Q/M428L mutant antibodies were performed by anonparametric Mann-Whitney test (two-tailed) using GraphPad Prism®,version 3.02 (Graphpad™ Software). P values less than 0.05 wereconsidered significant.

As shown in Table 17, the C_(max) was similar between the Dac-IgG1 WTand Dac-IgG1 T250Q/M428L groups, indicating that the administeredantibodies were distributed to the circulation in a similar manner.Thus, the higher antibody concentrations of the mutant IgG1 antibodyfollowing the distribution phase are attributable to its increasedpersistence in the serum. Analysis of the mean CL value indicated thatthis was the case. The mean CL was approximately 30% slower for theDac-IgG1 T250Q/M428L variant (0.169±0.020 ml/hr/kg; p=0.0002) comparedto Dac-IgG1 WT (0.243±0.018 ml/hr/kg) (Table 17), indicating asignificant decrease in the systemic clearance of the Dac-IgG1T250Q/M428L variant compared to the Dac-IgG1 WT antibody.

The PK profile of the Dac-IgG1 T250Q/M428L variant was further analyzedby calculating other parameters (Table 17). The mean AUC (extrapolatedto infinity) was approximately 45% higher for the Dac-IgG1 T250Q/M428Lvariant (59,800±7,000 hr*μg/ml; p=0.0002) compared to Dac-IgG1 WT(41,300±3,100 hr*μg/ml) (Table 17), indicating a significant increase inthe total exposure of the Dac-IgG1 T250Q/M428L variant compared to thewild-type antibody.

As a result of decreased clearance, the mean elimination (β-phase)half-life was approximately 30% longer for the Dac-IgG1 T250Q/M428Lvariant (240±32 hr; p=0.0052) compared to wild-type Dac-IgG1 (186±26 hr)(Table 17).

For the Dac-IgG2M3 T250Q/M428L variant antibody, however, the C_(max)appeared slightly lower compared to the Dac-IgG1 WT antibody, althoughthe difference was not statistically different (p=0.054). The mean CLwas approximately 15% slower for the Dac-IgG2M3 T250Q/M428L variant(0.208±0.067 ml/hr/kg) compared to Dac-IgG1 WT (0.243±0.018 ml/hr/kg)(Table 17). However, the decrease in the clearance of the Dac-IgG2M3T250Q/M428L variant was not significant compared to Dac-IgG1 WT(p=0.0728).

The mean AUC (extrapolated to infinity) was approximately 27% higher forthe Dac-IgG2M3 T250Q/M428L variant (52,400±16,900 hr*μg/ml) compared toDac-IgG1 WT (41,300±3,100 hr*μg/ml) (Table 17). However, the differencewas not significant due to the high inter-individual variability seen ingroup 3 (p=0.0728).

The mean elimination (β-phase) half-life was significantly(approximately 58%) longer for the Dac-IgG2M3 T250Q/M428L variant(293±140 hr; p=0.0111) compared to Dac-IgG1WT (186±26 hr) (Table 17).Note that the inter-animal variability is also quite high for thisparameter (CV % is approximately 48%).

TABLE 17 Elimination Name^(a) C_(max) ^(b) CL^(c) AUC^(d) half-life^(e)(Dac) (μg/ml) (ml/hr/kg) (hr * μg/ml) (hr) IgG1 WT 260 ± 27 0.243 ±0.018 41300 ± 3100 186 ± 26 IgG1 T250Q/M428L 278 ± 20 0.169* ± 0.020 59800* ± 7000  240* ± 32  IgG2M3 T250Q/M428L 220 ± 26 0.208 ± 0.067 52400 ± 16900 293* ± 140 ^(a)For each mutant, the first letterindicates the wild-type amino acid, the number indicates the positionaccording to the EU index (Kabat et al., op. cit.), and the secondletter indicates the mutant amino acid. ^(b)C_(max) values (±S.D.) areexpressed in μg/ml and were the observed maximum concentrations. ^(c)CLvalues (±S.D.) are expressed in ml/hr/kg and were calculated from the PKdata using WinNonlin as described in Example 15. ^(d)AUC values (±S.D.)are expressed in hr * μg/ml and were calculated from the PK data usingWinNonlin as described in Example 15. ^(e)Elimination half-life values(±S.D.) are expressed in hr and were calculated from the PK data usingWinNonlin as described in Example 15. *Indicates a significantdifference (p < 0.050) when comparing the wild-type group to theT250Q/M428L mutant group. Mann-Whitney tests were done using GraphPadPrism as described in Example 15.

Example 16

This example describes application of the various binding analysesdescribed in Examples 7 and 11 to mutants of the humanized anti-CD3antibody, visilizumab, with the IgG2M3 isotype.

Mutagenesis:

Amino acid substitutions at positions 250 and 428 of the visilizumab(Nuvion®; HuM291) IgG2M3 heavy chain (numbered according to the EU indexof Kabat et al., op. cit.) were generated following the methodsdescribed in Examples 3-4 above. The T250Q/M428L mutation wasincorporated into a HuM291-IgG2M3 heavy chain expression vector usingthe methods for vector design described in Examples 1-2. Wild-type lightchain and HuM291-IgG2M3 heavy chain expression vectors were alsoconstructed. The sequences of the T250Q/M428L variant of HuM291 aredepicted in FIG. 24 (SEQ ID NOS: 135 and 139).

Transfections:

293-H cells were transiently cotransfected with the HuM291 light chainplasmid and the HuM291-IgG2M3 WT or HuM291-IgG2M3 T250Q/M428L heavychain plasmids, as described in Example 5.

Antibody Purification:

Antibodies were purified from culture supernatants containingHuM291-IgG2M3 WT or HuM291-IgG2M3 T250Q/M428L by protein A affinitychromatography, following the methods described in Example 5.

SDS-PAGE:

Five μg samples of purified antibodies were run under reducing ornon-reducing conditions, as described in Example 5.

Competitive Binding Assays:

Antibodies were tested for binding to human FcRn on cell line NS0 HuFcRn(memb), clone 7-3, according to the methods described in Example 11.

pH-Dependent Binding and Release Assay:

The HuM291-IgG2M3 WT and HuM291-IgG2M3 T250Q/M428L mutant antibodieswere compared for binding to human FcRn and then released at various pHvalues in single-point binding and release assays using cell line NS0HuFcRn (memb), clone 7-3, as described in Example 7.

Results:

Amino acid substitutions were generated at positions 250 and 428(numbered according to the EU index of Kabat et al., op. cit.) of thehuman γ2M3 heavy chain of HuM291. These two positions were chosen basedon the identification of mutations at these positions in the human γ1and γ2M3 heavy chains of OST577 antibodies that resulted in increasedbinding affinities to FcRn and longer serum half-lives.

Purified antibodies were characterized by SDS-PAGE under non-reducingand reducing conditions. SDS-PAGE analysis under non-reducing conditionsindicated that the purified antibodies had a molecular weight of about150-160 kD (data not shown); analysis under reducing conditionsindicated that the purified antibodies were comprised of a heavy chainwith a molecular weight of about 50 kD and a light chain with amolecular weight of about 25 kD (data not shown).

The relative binding of the wild-type HuM291-IgG2M3 antibody and theHuM291-IgG2M3 T250Q/M428L double mutant to FcRn was determined using atransfected NS0 cell line stably expressing human FcRn on its surface.The purified antibodies were tested for FcRn binding in competitivebinding assays. Increasing concentrations of unlabeled competitorantibodies were incubated with cells in the presence of a sub-saturatingconcentration of biotinylated human IgG antibody (Sigma-Aldrich) in FBB,pH 6.0. As summarized in Table 18, the IC50 for the wild-typeHuM291-IgG2M3 antibody is ˜42 μg/ml, whereas the IC50 for theT250Q/M428L double mutant is ˜1.0 μg/ml

To confirm that binding was pH-dependent, the antibodies were tested forbinding to a transfected NS0 cell line stably expressing human FcRn andthen released at pH values ranging from pH 6.0 to pH 8.0. The cells wereincubated with a sub-saturating concentration of antibody in FBB, pH6.0, washed with FBB, pH 6.0, 6.5, 7.0, 7.5, or 8.0, and binding wasanalyzed by FACS™. The results indicated that wild-type HuM291-IgG2M3antibody and the modified HuM291-IgG2M3 antibody with the T250Q/M428Lmutation both showed strong binding to human FcRn at pH 6.0, withdiminishing binding as the pH values increased to pH 8.0. These resultsconfirmed that the binding of the HuM291-IgG2M3 antibodies to human FcRnwas pH-dependent.

TABLE 18 Name^(a) (IgG2M3) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 3 42.2 ± 7.5  1.0 T250Q/M428L 3 0.994 ± 0.200 42 ^(a)For eachmutant, the first letter indicates the wild-type amino acid, the numberindicates the position according to the EU index (Kabat et al., op.cit.), and the second letter indicates the mutant amino acid. ^(b)nindicates the number of independent assays. ^(c)IC50 values (±S.D.) areexpressed in μg/ml (based on final competitor concentrations) and werecalculated from competitive binding assays versus biotinylated human IgG(Sigma-Aldrich) in FBB, pH 6.0, as described in Example 16. ^(d)Relativebinding to human FcRn was calculated as the ratio of the IC50 value ofthe wild-type HuM291-IgG2M3 to that of the mutant.

Example 17

This example describes application of the various binding analysesdescribed in Examples 7 and 11 to mutants of the chimeric anti-α5β1integrin antibody, volociximab, with the IgG4 isotype.

Mutagenesis:

Amino acid substitutions at positions 250 and 428 of the volociximab(M200) IgG4 heavy chain (numbered according to the EU index of Kabat etal., op. cit.) were generated following the methods described inExamples 3-4 above. The T250Q/M428L mutation was incorporated into anM200-IgG4 heavy chain expression vector using the methods for vectordesign described in Examples 1-2. Wild-type light chain and M200-IgG4heavy chain expression vectors were also constructed. The sequences ofthe T250Q/M428L variant of M200 are depicted in FIG. 25 (SEQ ID NOS: 141and 145).

Transfections:

The M200-IgG4 WT and M200-IgG4 T250Q/M428L expression vectors werestably transfected into NS0 cells, as described in Example 6. Thehighest antibody-producing clones were chosen for expansion andadaptation to Protein-Free Basal Medium-2 (PFBM-2) (Protein DesignLabs™, Inc.). The cell lines were expanded to roller bottles in PFBM-2,supplemented after 2 days with 1/10 volume of PFFM-3, and grown toexhaustion.

Antibody Purification:

Antibodies were purified from culture supernatants containing M200-IgG4WT or M200-IgG4 T250Q/M428L by protein A affinity chromatography,following the methods described in Example 5.

SDS-PAGE:

Five μg samples of purified antibodies were run under reducing ornon-reducing conditions, as described in Example 5.

Competitive Binding Assays:

Antibodies were tested for binding to human FcRn on cell line NS0 HuFcRn(memb), clone 7-3, according to the methods described in Example 11.

pH-Dependent Binding and Release Assay:

The M200-IgG4 WT and M200-IgG4 T250Q/M428L mutant antibodies werecompared for binding to human FcRn and then released at various pHvalues in single-point binding and release assays using cell line NS0HuFcRn (memb), clone 7-3, as described in Example 7.

Results:

Amino acid substitutions were generated at positions 250 and 428(numbered according to the EU index of Kabat et al., op. cit.) of thehuman γ4 heavy chain of M200. These two positions were chosen based onthe identification of mutations at these positions in the human γ1 andγ2M3 heavy chains of OST577 antibodies that resulted in increasedbinding affinities to FcRn and longer serum half-lives.

Purified antibodies were characterized by SDS-PAGE under non-reducingand reducing conditions. SDS-PAGE analysis under non-reducing conditionsindicated that the purified antibodies had a molecular weight of about150-160 kD (data not shown); analysis under reducing conditionsindicated that the purified antibodies were comprised of a heavy chainwith a molecular weight of about 50 kD and a light chain with amolecular weight of about 25 kD (data not shown).

The relative binding of the wild-type M200-IgG4 antibody and theM200-IgG4 T250Q/M428L double mutant to FcRn was determined using atransfected NS0 cell line stably expressing human FcRn on its surface.The purified antibodies were tested for FcRn binding in competitivebinding assays. Increasing concentrations of unlabeled competitorantibodies were incubated with cells in the presence of a sub-saturatingconcentration of biotinylated human IgG antibody (Sigma-Aldrich) in FBB,pH 6.0. As summarized in Table 19, the IC50 for the wild-type M200-IgG4antibody is ˜35 μg/ml, whereas the IC50 for the T250Q/M428L doublemutant is ˜1.0 μg/ml

To confirm that binding was pH-dependent, the antibodies were tested forbinding to a transfected NS0 cell line stably expressing human FcRn andthen released at pH values ranging from pH 6.0 to pH 8.0. The cells wereincubated with a sub-saturating concentration of antibody in FBB, pH6.0, washed with FBB, pH 6.0, 6.5, 7.0, 7.5, or 8.0, and binding wasanalyzed by FACS™. The results indicated that wild-type M200-IgG4antibody and the modified M200-IgG4 antibody with the T250Q/M428Lmutation both showed strong binding to human FcRn at pH 6.0, withdiminishing binding as the pH values increased to pH 8.0 (data notshown). These results confirmed that the binding of the M200-IgG4antibodies to human FcRn was pH-dependent.

TABLE 19 Name^(a) (IgG4) n^(b) IC50 (μg/ml)^(c) Relative Binding^(d)Wild-type 3 34.7 ± 6.3  1.0 T250Q/M428L 3 1.00 ± 0.13 35 ^(a)For eachmutant, the first letter indicates the wild-type amino acid, the numberindicates the position according to the EU index (Kabat et al., op.cit.), and the second letter indicates the mutant amino acid. ^(b)nindicates the number of independent assays. ^(c)IC50 values (±S.D.) areexpressed in μg/ml (based on final competitor concentrations) and werecalculated from competitive binding assays versus biotinylated human IgG(Sigma-Aldrich) in FBB, pH 6.0, as described in Example 17. ^(d)Relativebinding to human FcRn was calculated as the ratio of the IC50 value ofthe wild-type M200-IgG4 to that of the mutant.

Although the invention has been described with reference to thepresently preferred embodiments, it should be understood that variousmodifications may be made without departing from the spirit of theinvention.

All publications, patents, patent applications, and web sites are hereinincorporated by reference in their entirety to the same extent as ifeach individual publication, patent, patent application, or web site wasspecifically and individually indicated to be incorporated by referencein its entirety.

1. A modified anti-CD25 monoclonal antibody of class IgG with FcRnbinding affinity increased relative to that of an unmodified monoclonalantibody of class IgG, the modified antibody comprising a heavy chainconstant region comprising glutamic acid or glutamine at amino acidresidue 250 and leucine or phenylalaine at amino acid residue
 428. 2.The modified monoclonal antibody according to claim 1, wherein saidunmodified monoclonal antibody is of IgG1 or IgG2M3 isotype.
 3. Themodified monoclonal antibody according to claim 1, wherein said classIgG antibody is a human IgG1.
 4. The modified monoclonal antibodyaccording to claim 1, wherein said class IgG antibody is a human IgG2M3.5. The modified monoclonal antibody according to claim 1, wherein saidamino acid residue 250 from the heavy chain constant region isglutamine.
 6. The modified monoclonal antibody according to claim 1,wherein said amino acid residue 250 from the heavy chain constant regionis glutamic acid.
 7. The modified monoclonal antibody according to claim2, wherein said amino acid residue 428 from the heavy chain constantregion is leucine.
 8. The modified monoclonal antibody according toclaim 2, wherein said amino acid residue 428 from the heavy chainconstant region is phenylalanine.